Long chain omega-3 and omega-6 polyunsaturated fatty acid biosynthesis by expression of acyl-CoA lysophospholipid acyltransferases

ABSTRACT

Methods for increasing C 18  to C 20  elongation conversion efficiency and/or Δ4 desaturation conversion efficiency in long-chain polyunsaturated fatty acid [“LC-PUFA”]-producing recombinant oleaginous microbial host cells are provided herein, based on over-expression of acyl-CoA:lysophospholipid acyltransferases [“LPLATs”] (e.g., Ale1, LPAAT, LPCAT). Production host cells and oils produced by the methods of the invention are also claimed.

This application is a divisional of application Ser. No. 12/814,764 (nowU.S. Pat. No. 8,524,485), filed Jun. 14, 2010, which claims the benefitof U.S. Provisional Application Nos. 61/187,366, 61/187,368 and61/187,359, each filed Jun. 16, 2009. The disclosures of all these priorapplications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to methods for increasing C₁₈ to C₂₀ elongationconversion efficiency and/or Δ4 desaturation conversion efficiency inlong-chain polyunsaturated fatty acid [“LC-PUFA”]-producing recombinantoleaginous microbial host cells, based on over-expression of genesencoding acyl-CoA:lysophospholipid acyltransferases [“LPLATs”].

BACKGROUND OF THE INVENTION

Glycerophospholipids, the main component of biological membranes,contain a glycerol core with fatty acids attached as R groups at thesn-1 position and sn-2 position, and a polar head group joined at thesn-3 position via a phosphodiester bond. The specific polar head group(e.g., phosphatidic acid, chloline, ethanolamine, glycerol, inositol,serine, cardiolipin) determines the name given to a particularglycerophospholipid, thus resulting in phosphatidylcholines [“PC”],phosphatidylethanolamines [“PE”], phosphatidylglycerols [“PG”],phosphatidylinositols [“PI”], phosphatidylserines [“PS”] andcardiolipins [“CL”]. Glycerophospholipids possess tremendous diversity,not only resulting from variable phosphoryl head groups, but also as aresult of differing chain lengths and degrees of saturation of theirfatty acids. Generally, saturated and monounsaturated fatty acids areesterified at the sn-1 position, while polyunsaturated fatty acids areesterified at the sn-2 position.

Glycerophospholipid biosynthesis is complex. Table 1 below summarizesthe steps in the de novo pathway, originally described by Kennedy andWeiss (J. Biol. Chem., 222:193-214 (1956)):

TABLE 1 General Reactions Of de Novo Glycerophospholipid Biosynthesissn-Glycerol-3-Phosphate → Glycerol-3-phosphate acyltransferase (GPAT)[E.C. Lysophosphatidic Acid 2.3.1.15] esterifies 1st acyl-CoA to sn-1position of (1-acyl-sn-glycerol 3- sn-glycerol 3-phosphate phosphate or“LPA”) LPA → Phosphatidic Acid Lysophosphatidic acid acyltransferase(LPAAT) [E.C. (1,2-diacylglycerol 2.3.1.51] esterifies 2nd acyl-CoA tosn-2 position of LPA phosphate or “PA”) PA → 1,2-DiacylglycerolPhosphatidic acid phosphatase [E.C.3.1.3.4] (“DAG”) removes a phosphatefrom PA; DAG can Or subsequently be converted to PC, PE or TAG (TAG PA →Cytidine Diphosphate synthesis requires either a diacylglycerolDiacylglycerol acyltransferase (DGAT) [E.C. 2.3.1.20] or a (“CDP-DG”)phospholipid: diacylglycerol acyltransferase (PDAT) [E.C.2.3.1.158])CDP-diacylglycerol synthase [EC 2.7.7.41] causes condensation of PA andcytidine triphosphate, with elimination of pyrophosphate; CDP-DG cansubsequently be converted to PI, PS, PG or CL

Following their de novo synthesis, glycerophospholipids can undergorapid turnover of the fatty acyl composition at the sn-2 position. This“remodeling”, or “acyl editing”, is important for membrane structure andfunction, biological response to stress conditions, and manipulation offatty acid composition and quantity in biotechnological applications.Specifically, the remodeling has been attributed to deacylation of theglycerophospholipid and subsequent reacylation of the resultinglysophospholipid.

In the Lands' cycle (Lands, W. E., J. Biol. Chem., 231:883-888 (1958)),remodeling occurs through the concerted action of: 1) a phospholipase,such as phospholipase A₂, that releases fatty acids from the sn-2position of phosphatidylcholine; and, 2) acyl-CoA:lysophospholipidacyltransferases [“LPLATs”], such as lysophosphatidylcholineacyltransferase [“LPCAT”] that reacylates the lysophosphatidylcholine[“LPC”] at the sn-2 position. Other glycerophospholipids can also beinvolved in the remodeling with their respective lysophospholipidacyltransferase activity, including LPLAT enzymes havinglysophosphatidylethanolamine acyltransferase [“LPEAT”] activity,lysophosphatidylserine acyltransferase [“LPLAT”] activity,lysophosphatidylglycerol acyltransferase [“LPGAT”] activity andlysophosphatidylinositol acyltransferase [“LPIAT”] activity. In allcases, LPLATs are responsible for removing acyl-CoA fatty acids from thecellular acyl-CoA pool and acylating various lysophospholipid substratesat the sn-2 position in the phospholipid pool. Finally, LPLATs alsoinclude LPAAT enzymes that are involved in the de novo biosynthesis ofPA from LPA. LPCAT activity is associated with two structurally distinctprotein families, wherein one belongs to the LPAAT family of proteinsand the other belongs to the membrane bound O-acyltransferase [“MBOAT”]family of proteins.

In other cases, this sn-2 position remodeling has been attributed to theforward and reverse reactions of enzymes having LPCAT activity (StymneS. and A. K. Stobart, Biochem J., 223(2):305-314 (1984)).

Several recent reviews by Shindou et al. provide an overview ofglycerophospholipid biosynthesis and the role of LPLATs (J. Biol. Chem.,284(1):1-5 (2009); J. Lipid Res., 50:S46-S51 (2009)). Numerous LPLATshave been reported in public and patent literature, based on a varietyof conserved motifs.

The effect of LPLATs on polyunsaturated fatty acid [“PUFA”] productionhas also been contemplated, since fatty acid biosynthesis requires rapidexchange of acyl groups between the acyl-CoA pool and the phospholipidpool. Specifically, desaturations occur mainly at the sn-2 position ofphospholipids, while elongation occurs in the acyl-CoA pool. Forexample, Intl. App. Pub. No. WO 2004/076617 describes the isolation ofan LPCAT from Caenorhabditis elegans (clone T06E8.1) and reportsincrease in the efficiency of Δ6 desaturation and Δ6 elongation, as wellas an increase in biosynthesis of the long-chain PUFAs eicosadienoicacid [“EDA”; 20:2] and eicosatetraenoic acid [“ETA”; 20:4],respectively, when the LPCAT was expressed in an engineered strain ofSaccharomyces cerevisiae that was fed exogenous 18:2 or α-linolenic[“ALA”; 18:3] fatty acids, respectively.

Furthermore, Example 16 of Intl. App. Pub. No. WO 2004/087902 describesthe isolation of Mortierella alpina LPAAT-like proteins (encoded by theproteins of SEQ ID NO:93 and SEQ ID NO:95, having 417 amino acids inlength or 389 amino acids in length, respectively) that are identicalexcept for an N-terminal extension of 28 amino acid residues in SEQ IDNO:93. Intl. App. Pub. No. WO 2004/087902 also reports expression of oneof these proteins using similar methods to those of Intl. App. Pub. No.WO 2004/076617, which results in similar improvements in EDA and ETAbiosynthesis.

Both Intl. App. Publications No. WO 2004/076617 and No. WO 2004/087902teach that the improvements in EDA and ETA biosynthesis are due toreversible LPCAT activity in some LPAAT-like proteins, although not allLPAAT-like proteins have LPCAT activity. They do not teach that LPCATexpression would result in the improvements in strains that do notrequire exogenous feeding of fatty acid substrates or in microbialspecies other than Saccharomyces cerevisiae. They also do not teach thatLPCAT expression in engineered microbes results in increased productionof high LC-PUFAs other than EDA and ETA, such as ARA, EPA and DHA, orthat LPCAT expression can result in improvement in alternatedesaturation reactions, other than Δ6 desaturation. Neither referenceteaches the effect of the LPCAT or LPAAT-like proteins on either Δ6elongation without exogenous feeding of fatty acids or on Δ4desaturation.

Numerous other references generally describe benefits of co-expressingLPLATs with PUFA biosynthetic genes, to increase the amount of a desiredfatty acid in the oil of a transgenic organism, increase total oilcontent or selectively increase the content of desired fatty acids(e.g., Intl. App. Publications No. WO 2004/087902, No. WO 2006/069936,No. WO 2006/052870, No. WO 2009/001315, No. WO 2009/014140).

Despite the work describe above, to date no one has studied the effectof LPAATs and LPCATs in an oleaginous organism engineered for high-levelproduction of LC-PUFAs other than EDA and ETA, such as eicosapentaenoicacid [“EPA”; cis-5,8,11,14,17-eicosapentaenoic acid] and/ordocosahexaenoic acid [“DHA”; cis-4,7,10,13,16,19-docosahexaenoic acid]and for improved C₁₈ to C₂₀ elongation conversion efficiency, and/orimproved Δ4 desaturation conversion efficiency without exogenouslyfeeding fatty acids.

SUMMARY OF THE INVENTION

In one embodiment, the invention concerns a recombinant oleaginousmicrobial host cell for the improved production of at least onelong-chain polyunsaturated fatty acid, said host cell comprising atleast one isolated polynucleotide encoding a polypeptide having at leastacyl-CoA:lysophospholipid acyltransferase activity wherein thepolypeptide is selected from the group consisting of:

-   -   (i) a polypeptide having at least 45% amino acid identity, based        on the Clustal W method of alignment, when compared to an amino        acid sequence selected from the group consisting of SEQ ID NO:9        and SEQ ID NO:11;    -   (ii) a polypeptide having at least one membrane bound        O-acyltransferase protein family motif selected from the group        consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID        NO:28;    -   (iii) a polypeptide having at least 90% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence as set forth in SEQ ID NO:2;    -   (iv) a polypeptide having at least 43.9% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence selected from the group consisting of SEQ ID        NO:15, SEQ ID NO:17 and SEQ ID NO:18; and,    -   (v) a polypeptide having at least one        1-acyl-sn-glycerol-3-phosphate acyltransferase family motif        selected from the group consisting of: SEQ ID NO:19 and SEQ ID        NO:20;

wherein the at least one isolated polynucleotide encoding a polypeptidehaving at least acyl-CoA:lysophospholipid acyltransferase activity isoperably linked to at least one regulatory sequence, said regulatorysequence being the same or different, and

further wherein the host cell has at least one improvement selected fromthe group consisting of:

a) an increase in C₁₈ to C₂₀ elongation conversion efficiency in atleast one long-chain polyunsaturated fatty acid-producing oleaginousmicrobial host cell when compared to a control host cell;

b) an increase in Δ4 desaturation conversion efficiency in at least onelong-chain polyunsaturated fatty acid-producing oleaginous microbialhost cell when compared to a control host cell.

The recombinant oleaginous microbial host cell can be yeast, preferably,Yarrowia lipolytica.

In a second embodiment, the invention concerns a recombinant oleaginousmicrobial host cell for the improved production of at least onelong-chain polyunsaturated fatty acid wherein the long-chainpolyunsaturated fatty acid can be selected from the group consisting of:eicosadienoic acid, dihomo-γ-linolenic acid, arachidonic acid,eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid,docosatetraenoic acid, ω-6 docosapentaenoic acid, ω-3 docosapentaenoicacid and docosahexaenoic acid.

In a third embodiment, the invention concerns a recombinant oleaginousmicrobial host cell for the improved production of at least onelong-chain polyunsaturated fatty acid wherein the polynucleotideencoding a polypeptide having at least acyl-CoA:lysophospholipidacyltransferase activity is stably integrated; and, further wherein thehost cell has at least one improvement selected from the groupconsisting of:

a) an increase in C₁₈ to C₂₀ elongation conversion efficiency of atleast 4% in at least one long-chain polyunsaturated fatty acid-producingoleaginous microbial host cell when compared to a control host cell;and,

b) an increase in Δ4 desaturation conversion efficiency of at least 5%in at least one long-chain polyunsaturated fatty acid-producingoleaginous microbial host cell when compared to a control host cell.

In a fourth embodiment, the improvement in production of at least onelong-chain polyunsaturated fatty acid can be selected from the groupconsisting of:

a) an increase in C₁₈ to C₂₀ elongation conversion efficiency of atleast 13% in an eicosapentaenoic acid-producing host cell when comparedto a control host cell;

b) an increase in C₁₈ to C₂₀ elongation conversion efficiency of atleast 4% in a docosahexaenoic acid-producing host cell when compared toa control host cell;

c) an increase in Δ4 desaturation conversion efficiency of at least 18%in a docosahexaenoic acid-producing host cell when compared to a controlhost cell;

d) an increase of at least 9 weight percent of eicosapentaenoic acid inan eicosapentaenoic acid-producing host cell measured as a weightpercent of the total fatty acids when compared to a control host cell;

e) an increase of at least 2 weight percent of eicosapentaenoic acid ina docosahexaenoic acid-producing host cell measured as a weight percentof the total fatty acids when compared to a control host cell; and,

f) an increase of at least 9 weight percent of docosahexaenoic acid in adocosahexaenoic acid-producing host cell measured as a weight percent ofthe total fatty acids when compared to a control host cell.

In a fifth embodiment, the invention concerns oil comprisingeicosapentaenoic acid and/or docosahexaenoic acid obtained from theoleaginous microbial recombinant host cell of the invention.

In a sixth embodiment, the invention concerns a method for making an oilcomprising eicosapentaenoic acid and/or docosahexaenoic acid comprising:

a) culturing the oleaginous microbial host cell of claim 3 wherein anoil comprising eicosapentaenoic acid and/or docosahexaenoic acid isproduced; and,

b) optionally recovering the microbial oil of step (a).

In a seventh embodiment, the invention concerns a method for increasingC₁₈ to C₂₀ elongation conversion efficiency in a long-chainpolyunsaturated fatty acid-producing oleaginous microbial recombinanthost cell, comprising:

a) introducing into said long-chain polyunsaturated fatty acid-producingrecombinant host cell at least one isolated polynucleotide encoding apolypeptide having at least acyl-CoA:lysophospholipid acyltransferaseactivity wherein the polypeptide is selected from the group consistingof:

-   -   (i) a polypeptide having at least 45% amino acid identity, based        on the Clustal W method of alignment, when compared to an amino        acid sequence selected from the group consisting of SEQ ID NO:9        and SEQ ID NO:11;    -   (ii) a polypeptide having at least one membrane bound        O-acyltransferase protein family motif selected from the group        consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID        NO:28;    -   (iii) a polypeptide having at least 90% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence as set forth in SEQ ID NO:2;    -   (iv) a polypeptide having at least 43.9% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence selected from the group consisting of SEQ ID        NO:15, SEQ ID NO:17 and SEQ ID NO:18; and,    -   (v) a polypeptide having at least one        1-acyl-sn-glycerol-3-phosphate acyltransferase protein family        motif selected from the group consisting of: SEQ ID NO:19 and        SEQ ID NO:20;

wherein the at least one isolated polynucleotide encoding a polypeptidehaving at least acyl-CoA:lysophospholipid acyltransferase activity isoperably linked to at least one regulatory sequence, said regulatorysequence being the same or different; and,

b) growing the oleaginous microbial host cell;

wherein the C₁₈ to C₂₀ elongation conversion efficiency of theoleaginous microbial host cell is increased relative to the control hostcell.

In a eighth embodiment, the invention concerns a method of the inventionwherein:

-   -   a) the polynucleotide encoding a polypeptide having at least        acyl-CoA:lysophospholipid acyltransferase activity is stably        integrated; and,    -   b) the increase in C₁₈ to C₂₀ elongation conversion efficiency        is at least 13% in an eicosapentaenoic acid-producing host cell        when compared to the control host cell and/or the increase in        C₁₈ to C₂₀ elongation conversion efficiency is at least 4% in a        docosahexaenoic acid-producing host cell when compared to the        control host cell.

In an ninth embodiment, the invention concerns a method for increasingΔ4 desaturation conversion efficiency in a long-chain polyunsaturatedfatty acid-producing oleaginous microbial recombinant host cell,comprising:

a) introducing into said long-chain polyunsaturated fatty acid-producingrecombinant host cell at least one isolated polynucleotide encoding apolypeptide having at least acyl-CoA:lysophospholipid acyltransferaseactivity wherein the polypeptide is selected from the group consistingof:

-   -   (i) a polypeptide having at least 45% amino acid identity, based        on the Clustal W method of alignment, when compared to an amino        acid sequence selected from the group consisting of SEQ ID NO:9        and SEQ ID NO:11;    -   (ii) a polypeptide having at least one membrane bound        O-acyltransferase protein family motif selected from the group        consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID        NO:28;    -   (iii) a polypeptide having at least 90% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence as set forth in SEQ ID NO:2;    -   (iv) a polypeptide having at least 43.9% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence selected from the group consisting of SEQ ID        NO:15, SEQ ID NO:17 and SEQ ID NO:18; and,    -   (v) a polypeptide having at least one        1-acyl-sn-glycerol-3-phosphate acyltransferase protein family        motif selected from the group consisting of: SEQ ID NO:19 and        SEQ ID NO:20;    -   wherein the at least one isolated polynucleotide encoding a        polypeptide having at least acyl-CoA:lysophospholipid        acyltransferase activity is operably linked to at least one        regulatory sequence, said regulatory sequence being the same or        different, and,

b) growing the oleaginous microbial host cell;

wherein the Δ4 desaturation conversion efficiency of the oleaginousmicrobial host cell is increased relative to the control host cell.

In a tenth embodiment, the invention concerns a method for increasing Δ4desaturation conversion efficiency in a long-chain polyunsaturated fattyacid-producing oleaginous microbial recombinant host cell wherein:

-   -   a) the polynucleotide encoding a polypeptide having at least        acyl-CoA:lysophospholipid acyltransferase activity is stably        integrated; and,    -   b) the increase in Δ4 desaturation conversion efficiency is at        least 18% when compared to a control host cell.

BIOLOGICAL DEPOSITS

The following biological materials have been deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, and bear the following designations, accession numbersand dates of deposit.

Biological Material Accession No. Date of Deposit Yarrowia lipolyticaY4128 ATCC PTA-8614 Aug. 23, 2007 Yarrowia lipolytica Y8406 ATCCPTA-10025 May 14, 2009 Yarrowia lipolytica Y8412 ATCC PTA-10026 May 14,2009

The biological materials listed above were deposited under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. The listed depositwill be maintained in the indicated international depository for atleast 30 years and will be made available to the public upon the grantof a patent disclosing it. The availability of a deposit does notconstitute a license to practice the subject invention in derogation ofpatent rights granted by government action.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1A and FIG. 1B illustrate the ω-3/ω-6 fatty acid biosyntheticpathway, and should be viewed together when considering the descriptionof this pathway.

FIG. 2 diagrams the development of Yarrowia lipolytica strain Y8406,producing greater than 51.2 EPA % TFAs.

FIG. 3 provides a plasmid map for pY116.

FIG. 4A provides a plasmid map for pZKSL-5S5A5; and FIG. 4B provides aplasmid map for pZP3-Pa777U.

FIG. 5A provides a plasmid map for pZKUM; and FIG. 5B provides a plasmidmap for pZKL2-5 mB89C.

FIG. 6A provides a plasmid map for pZKL1-2SR9G85; and FIG. 6B provides aplasmid map for pZSCP-Ma83.

FIG. 7 diagrams the development of Yarrowia lipolytica strain Y5037,producing 18.6 EPA % TFAs, 22.8 DPA % TFAs and 9.7 DHA % TFAs.

FIG. 8A provides a plasmid map for pZKL4-220EA41B; and FIG. 8B providesa plasmid map for pZKL3-4GER44.

FIG. 9 provides a plasmid map for pZKLY-G20444.

FIG. 10A provides a plasmid map for pY201, comprising a chimericYAT1::ScAle1S::Lip1 gene; and FIG. 10B provides a plasmid map for pY168,comprising a chimeric YAT1::YlAle1::Lip1 gene.

FIG. 11A provides a plasmid map for pY208, comprising a chimericYAT1::MaLPAAT1S::Lip1 gene; and FIG. 11B provides a plasmid map forpY207, comprising a chimeric YAT1::YlLPAAT1::Lip1 gene.

FIG. 12A provides a plasmid map for pY175, comprising a chimericYAT1::CeLPCATS::Lip1 gene; and FIG. 12B provides a plasmid map forpY153, comprising a chimeric FBAIN::CeLPCATS::YlLPAAT1 gene.

FIG. 13A provides a plasmid map for pY222, comprising a chimericYAT1::ScLPAATS::Lip1 gene; and FIG. 13B provides a plasmid map forpY177, comprising a chimeric YAT1::YlLPAAT1::Lip1 gene.

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for patent applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1-101 are ORFs encoding promoters, genes or proteins (orfragments thereof) or plasmids, as identified in Table 2.

TABLE 2 Summary of Gene and Protein SEQ ID Numbers Nucleic acid ProteinDescription SEQ ID NO. SEQ ID NO. Caenorhabditis elegans LPCAT(“CeLPCAT”)  1  2  (849 bp) (282 AA) membrane bound O-acyltransferasemotif —  3 M(V/I)LxxKL membrane bound O-acyltransferase motif —  4RxKYYxxW membrane bound O-acyltransferase motif SAxWHG —  5 SyntheticLPCAT derived from Caenorhabditis  6  7 elegans, codon-optimized forexpression in Yarrowia  (859 bp) (282 AA) lipolytica (“CeLPCATS”)Saccharomyces cerevisiae Ale1 (“ScAle1”; also ORF  8  9 “YOR175C”) (1860bp) (619 AA) Yarrowia lipolytica Ale1 (“YlAle1”) 10 11 (1539 bp) (512AA) Synthetic Ale1 derived from Saccharomyces 12 13 cerevisiae,codon-optimized for expression in (1870 bp) (619 AA) Yarrowia lipolytica(“ScAle1S”) Mortierella alpina LPAAT1 (“MaLPAAT1”) 14 15  (945 bp) (314AA) Yarrowia lipolytica LPAAT1 (“YlLPAAT1”) 16 17 (1549 bp) (282 AA)Saccharomyces cerevisiae LPAAT (“ScLPAAT”; also — 18 ORF “YDL052C”) (303AA) 1-acyl-sn-glycerol-3-phosphate acyltransferase motif — 19 NHxxxxD1-acyl-sn-glycerol-3-phosphate acyltransferase motif — 20 EGTR SyntheticLPAAT1 derived from Mortierella alpina, 21 22 codon-optimized forexpression in Yarrowia lipolytica  (955 bp) (314 AA) (“MaLPAAT1S”)Shindou et al. membrane bound O-acyltransferase — 23 motif WHGxxxGYxxxFShindou et al. membrane bound O-acyltransferase — 24 motif YxxxxFShindou et al. membrane bound O-acyltransferase — 25 motif YxxxYFxxHU.S. patent Pub. No. 2008-0145867-A1 motif — 26M-[V/I]-[L/I]-xxK-[L/V/I]-xxxxxxDG U.S. patent Pub. No. 2008-0145867-A1motif — 27 RxKYYxxWxxx-[E/D]-[A/G]xxxxGxG-[F/Y]-xG U.S. patent Pub. No.2008-0145867-A1 motif — 28 EX₁₁WNX₂-[T/V]-X₂W U.S. patent Pub. No.2008-0145867-A1 motif — 29 SAxWHGxxPGYxx-[T/F]-F Lewin, T. W. et al. &Yamashita et al. 1-acyl-sn- — 30 glycerol-3-phosphate acyltransferasemotif GxxFI-[D/R]-R Lewin, T. W. et al. 1-acyl-sn-glycerol-3-phosphate —31 acyltransferase motif [V/I]-[P/X]-[I/V/L]-[I/V]-P-[V/I] Yamashita etal. 1-acyl-sn-glycerol-3-phosphate — 32 acyltransferase motif IVPIVMPlasmid pY116 33 — (8739 bp) Plasmid pZKSL-5S5A5 34 — (13,975 bp)  Synthetic mutant Δ5 desaturase (“EgD5SM”), 35 36 derived from Euglenagracilis (“EgD5S”) (U.S. patent (1350 bp) (449 AA) Pub. No.2010-0075386-A1) Synthetic mutant Δ5 desaturase (“EaD5SM”), 37 38derived from Euglena anabaena (“EaD5S”) (U.S. (1365 bp) (454 AA) patentPub. No. 2010-0075386-A1) Plasmid pZP3-Pa777U 39 — (13,066 bp)   PlasmjdpZKUM 40 — (4313 bp) Plasmid pZKL2-5mB89C 41 — (15,991 bp)   Yarrowialipolytica diacylglycerol 42 43 cholinephosphotransferase gene(“YlCPT1”) (1185 bp) (394 AA) Synthetic mutant Δ8 desaturase (“EgD8M”)(U.S. 44 45 patent No. 7,709,239), derived from Euglena gracilis (1272bp) (422 AA) (“EgD8S”) (U.S. Pat. No. 7,256,033) Synthetic Δ9 elongasederived from Euglena gracilis, 46 47 codon-optimized for expression inYarrowia lipolytica  (777 bp) (258 AA) (“EgD9eS”) Plasmid pZKL1-2SR9G8548 — (14,554 bp)   DGLA synthase, comprising E389D9eS/EgD8M gene 49 50fusion (2127 bp) (708 AA) Synthetic Δ12 desaturase derived from Fusarium51 52 moniliforme, codon-optimized for expression in (1434 bp) (477 AA)Yarrowia lipolytica (“FmD12S”) Plasmid pZSCP-Ma83 53 — (15,119 bp)  Synthetic C_(16/18) elongase derived from Mortierella 54 55 alpina ELO3,codon-optimized for expression in  (828 bp) (275 AA) Yarrowia lipolytica(“ME3S”) Synthetic malonyl-CoA synthetase derived from 56 57 Rhizobiumleguminosarum bv. viciae 3841 (GenBank (1518 bp) (505 AA) Accession No.YP_766603), codon-optimized for expression in Yarrowia lipolytica(“MCS”) Synthetic Δ8 desaturase derived from Euglena 58 59 anabaena UTEX373, codon-optimized for (1260 bp) (420 AA) expression in Yarrowialipolytica (“EaD8S”) Plasmid pZKL4-220EA41B 60 — (16,424 bp)   SyntheticC20 elongase derived from Euglena 61 62 anabaena, codon-optimized forexpression in  (900 bp) (299 AA) Yarrowia lipolytica (“EaC20ES”)Synthetic C20 elongase derived from Euglena 63 64 gracilis,codon-optimized for expression in Yarrowia  (912 bp) (303 AA) lipolytica(“EgC20ES”) Truncated synthetic Δ4 desaturase derived from 65 66 Euglenaanabaena, codon-optimized for expression (1644 bp) (547 AA) in Yarrowialipolytica (“EaD4S-1”) Truncated synthetic Δ4 desaturase version Bderived 67 68 from Euglena anabaena, codon-optimized for (1644 bp) (547AA) expression in Yarrowia lipolytica (“EaD4SB”) Plasmid pZKL3-4GER44 69— (17,088 bp)   Synthetic Δ4 desaturase derived from Eutreptiella 70 71cf_gymnastica CCMP1594, codon-optimized for (1548 bp) (515 AA)expression in Yarrowia lipolytica (“E1594D4S”) Truncated synthetic Δ4desaturase derived from 72 73 Euglena gracilis, codon-optimized forexpression in (1542 bp) (513 AA) Yarrowia lipolytica (“EgD4S-1”) PlasmidpZKLY-G20444 74 — (15,617 bp)   Synthetic DHA synthase derived fromEuglena 75 76 gracilis, codon-optimized for expression in Yarrowia (2382bp) (793 AA) lipolytica (“EgDHAsyn1S”) Plasmid pY201 77 — (9641 bp)Escherichia coli LoxP recombination site, recognized 78 — by a Crerecombinase enzyme  (34 bp) Primer 798 79 — Primer 799 80 — Primer 80081 — Primer 801 82 — Plasmid pY168 83 — (9320 bp) Plasmid pY208 84 —(8726 bp) Primer 856 85 — Primer 857 86 — Plasmid pY207 87 — (8630 bp)Plasmid pY175 88 — (8630 bp) Plasmid pY153 89 — (8237 bp) Mutant Δ5desaturase (“EgD5M”), derived from 90 91 Euglena gracilis (“EgD5”) (U.S.patent Pub. No. (1350 bp) (449 AA) 2010-0075386-A1) Mortierella alpinaLPAAT (corresponding to SEQ ID 92 93 NOs: 16 and 17 within Intl. App.Pub. No. WO (1254 bp) (417 AA) 2004/087902) Mortierella alpina LPAAT(corresponding to SEQ ID 94 95 NOs: 18 and 19 within Intl. App. Pub. No.WO (1170 bp) (389 AA) 2004/087902) Synthetic LPAAT derived fromSaccharomyces 96 97 cerevisiae, codon-optimized for expression in  (926bp) (303 AA) Yarrowia lipolytica (“ScLPAATS”) Primer 869 98 — Primer 87099 — Plasmid pY222 100  — (7891 bp) Plasmid pY177 101  — (9598 bp)

DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods for increasing C₁₈ to C₂₀ elongationconversion efficiency and/or Δ4 desaturation conversion efficiency inlong-chain polyunsaturated fatty acid [“LC-PUFA”]-producing recombinantoleaginous microbial host cells, based on expression of polypeptides(e.g., Ale1, LPAAT, and LPCAT) having LPLAT activity. By increasing theconversion efficiency of C₁₈ to C₂₀ elongation and/or Δ4 desaturation,the concentration of the LC-PUFAs eicosapentaenoic acid [“EPA”;cis-5,8,11,14,17-eicosapentaenoic acid] and/or docosahexaenoic acid[“DHA”; cis-4,7,10,13,16,19-docosahexaenoic acid] increased as a weightpercent of the total fatty acids. Recombinant host cells are alsoclaimed.

PUFAs, such as EPA and DHA (or derivatives thereof), are used as dietarysubstitutes, or supplements, particularly infant formulas, for patientsundergoing intravenous feeding or for preventing or treatingmalnutrition. Alternatively, the purified PUFAs (or derivatives thereof)may be incorporated into cooking oils, fats or margarines formulated sothat in normal use the recipient would receive the desired amount fordietary supplementation. The PUFAs may also be incorporated into infantformulas, nutritional supplements or other food and drink products andmay find use as cardiovascular-protective, anti-depression,anti-inflammatory or cholesterol lowering agents. Optionally, thecompositions may be used for pharmaceutical use, either human orveterinary.

All patents, patent applications, and publications cited herein areincorporated by reference in their entirety.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

“Open reading frame” is abbreviated as “ORF”.

“Polymerase chain reaction” is abbreviated as “PCR”.

“American Type Culture Collection” is abbreviated as “ATCC”.

“Polyunsaturated fatty acid(s)” is abbreviated as “PUFA(s)”.

“Diacylglycerol acyltransferase” is abbreviated as “DAG AT” or “DGAT”.

“Triacylglycerols” are abbreviated as “TAGs”.

“Co-enzyme A” is abbreviated as “CoA”.

“Total fatty acids” are abbreviated as “TFAs”.

“Fatty acid methyl esters” are abbreviated as “FAMEs”.

“Dry cell weight” is abbreviated as “DCW”.

“Long-chain polyunsaturated fatty acid(s)” is abbreviated as“LC-PUFA(s)”.

“Acyl-CoA:lysophospholipid acyltransferase(s)” or “lysophospholipidacyltransferase(s)” is abbreviated as “LPLAT(s)”.

The term “invention” or “present invention” as used herein is not meantto be limiting to any one specific embodiment of the invention butapplies generally to any and all embodiments of the invention asdescribed in the claims and specification.

The term “glycerophospholipids” refers to a broad class of molecules,having a glycerol core with fatty acids at the sn-1 position and sn-2position, and a polar head group (e.g., phosphate, choline,ethanolamine, glycerol, inositol, serine, cardiolipin) joined at thesn-3 position via a phosphodiester bond. Glycerophospholipids thusinclude phosphatidylcholines [“PC”], phosphatidylethanolamines [“PE”],phosphatidylglycerols [“PG”], phosphatidylinositols [“PI”],phosphatidylserines [“PS”] and cardiolipins [“CL”].

“Lysophospholipids” are derived from glycerophospholipids, bydeacylation of the sn-2 position fatty acid. Lysophospholipids include,e.g., lysophosphatidic acid [“LPA”], lysophosphatidylcholine [“LPC”],lysophosphatidyletanolamine [“LPE”], lysophosphatidylserine [“LPS”],lysophosphatidylglycerol [“LPG”] and lysophosphatidylinositol [“LPI”].

The term “acyltransferase” refers to an enzyme responsible fortransferring an acyl group from a donor lipid to an acceptor lipidmolecule.

The term “acyl-CoA:lysophospholipid acyltransferase” or“lysophospholipid acyltransferase” [“LPLAT”] refers to a broad class ofacyltransferases, having the ability to acylate a variety oflysophospholipid substrates at the sn-2 position. More specifically,LPLATs include LPA acyltransferases [“LPAATs”] having the ability tocatalyze conversion of LPA to PA, LPC acyltransferases [“LPCATs”] havingthe ability to catalyze conversion of LPC to PC, LPE acyltransferases[“LPEATs”] having the ability to catalyze conversion of LPE to PE, LPSacyltransferases [“LPLATs”] having the ability to catalyze conversion ofLPS to PS, LPG acyltransferases [“LPGATs”] having the ability tocatalyze conversion of LPG to PG, and LPI acyltransferases [“LPIATs”]having the ability to catalyze conversion of LPI to PI. Standardizationof LPLAT nomenclature has not been formalized, so various otherdesignations are used in the art (for example, LPAATs have also beenreferred to as acyl-CoA:1-acyl-sn-glycerol-3-phosphate2-O-acyltransferases, 1-acyl-sn-glycerol-3-phosphate acyltransferasesand/or 1-acylglycerolphosphate acyltransferases [“AGPATs”] and LPCATsare often referred to as acyl-CoA:1-acyl lysophosphatidyl-cholineacyltransferases). Additionally, it is important to note that someLPLATs, such as the Saccharomyces cerevisiae Ale1 (ORF YOR175C; SEQ IDNO:9), have broad specificity and thus a single enzyme may be capable ofcatalyzing several LPLAT reactions, including LPAAT, LPCAT and LPEATreactions (Tamaki, H. et al., J. Biol. Chem., 282:34288-34298 (2007);Stahl, U. et al., FEBS Letters, 582:305-309 (2008); Chen, Q. et al.,FEBS Letters, 581:5511-5516 (2007); Benghezal, M. et al., J. Biol.Chem., 282:30845-30855 (2007); Riekhof, et al., J. Biol. Chem.,282:28344-28352 (2007)).

More specifically, the term “polypeptide having at leastlysophosphtidylcholine acyltransferase [“LPCAT”] activity” will refer tothose enzymes capable of catalyzing the reaction:acyl-CoA+1-acyl-sn-glycero-3-phosphocholine=CoA+1,2-diacyl-sn-glycero-3-phosphocholine(EC 2.3.1.23). LPCAT activity has been described in two structurallydistinct protein families, i.e., the LPAAT protein family (Hishikawa, etal., Proc. Natl. Acad. Sci. U.S.A., 105:2830-2835 (2008); Intl. App.Pub. No. WO 2004/076617) and the ALE1 protein family (Tamaki, H. et al.,supra; Stahl, U. et al., supra; Chen, Q. et al., supra; Benghezal, M. etal., supra; Riekhof, et al., supra).

The term “LPCAT” refers to a protein of the ALE1 protein family that: 1)has LPCAT activity (EC 2.3.1.23) and shares at least about 45% aminoacid identity, based on the Clustal W method of alignment, when comparedto an amino acid sequence selected from the group consisting of SEQ IDNO:9 (ScAle1) and SEQ ID NO:11 (YlAle1); and/or, 2) has LPCAT activity(EC 2.3.1.23) and has at least one membrane bound O-acyltransferase[“MBOAT”] protein family motif selected from the group consisting of:M(V/I)LxxKL (SEQ ID NO:3), RxKYYxxW (SEQ ID NO:4), SAxWHG (SEQ ID NO:5)and EX₁₁WNX₂— [T/V]-X₂W (SEQ ID NO:28). Examples of ALE1 polypeptidesinclude ScAle1 and YlAle1.

The term “ScAle1” refers to a LPCAT (SEQ ID NO:9) isolated fromSaccharomyces cerevisiae (ORF “YOR175C”), encoded by the nucleotidesequence set forth as SEQ ID NO:8. In contrast, the term “ScAle1S”refers to a synthetic LPCAT derived from S. cerevisiae that iscodon-optimized for expression in Yarrowia lipolytica (i.e., SEQ IDNOs:12 and 13).

The term “YlAle1” refers to a LPCAT (SEQ ID NO:11) isolated fromYarrowia lipolytica, encoded by the nucleotide sequence set forth as SEQID NO:10.

The term “LPCAT” also refers to a protein that has LPCAT activity (EC2.3.1.23) and shares at least about 90% amino acid identity, based onthe Clustal W method of alignment, when compared to an amino acidsequence as set forth in SEQ ID NO:2 (CeLPCAT).

The term “CeLPCAT” refers to a LPCAT enzyme (SEQ ID NO:2) isolated fromCaenorhabditis elegans, encoded by the nucleotide sequence set forth asSEQ ID NO:1. In contrast, the term “CeLPCATS” refers to a syntheticLPCAT derived from C. elegans that is codon-optimized for expression inYarrowia lipolytica (i.e., SEQ ID NOs:6 and 7).

The term “polypeptide having at least lysophosphatidic acidacyltransferase [“LPAAT”] activity” will refer to those enzymes capableof catalyzing the reaction: acyl-CoA+1-acyl-sn-glycerol3-phosphate=CoA+1,2-diacyl-sn-glycerol 3-phosphate (EC 2.3.1.51).

The term “LPAAT” refers to a protein that: 1) has LPAAT activity andshares at least about 43.9% amino acid identity, based on the Clustal Wmethod of alignment, when compared to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:15 (MaLPAAT1), SEQ ID NO:17(YlLPAAT1) and SEQ ID NO:18 (ScLPAAT1); and/or, 2) has LPAAT activityand has at least one 1-acyl-sn-glycerol-3-phosphate acyltransferasefamily motif selected from the group consisting of: NHxxxxD (SEQ IDNO:19) and EGTR (SEQ ID NO:20). Examples of LPAAT polypeptides includeScLPAAT, MaLPAAT1 and YlLPAAT1.

The term “ScLPAAT” refers to a LPAAT (SEQ ID NO:18) isolated fromSaccharomyces cerevisiae (ORF “YDL052C”).

The term “MaLPAAT1” refers to a LPAAT (SEQ ID NO:15) isolated fromMortierella alpina, encoded by the nucleotide sequence set forth as SEQID NO:14. In contrast, the term “MaLPAAT1S” refers to a synthetic LPAATderived from M. alpina that is codon-optimized for expression inYarrowia lipolytica (i.e., SEQ ID NOs:21 and 22).

The term “YlLPAAT1” refers to a LPAAT (SEQ ID NO:17) isolated fromYarrowia lipolytica, encoded by the nucleotide sequence set forth as SEQID NO:16.

The term “ortholog” refers to a homologous protein from a differentspecies that evolved from a common ancestor protein as evidenced bybeing in one clade of phylogenetic tree analysis and that catalyzes thesame enzymatic reaction.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions likely indicate amino acids that areessential in the structure, the stability, or the activity of a protein.Because they are identified by their high degree of conservation inaligned sequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family.

The term “oil” refers to a lipid substance that is liquid at 25° C. andusually polyunsaturated. In oleaginous organisms, oil constitutes amajor part of the total lipid. “Oil” is composed primarily oftriacylglycerols [“TAGs”] but may also contain other neutral lipids,phospholipids and free fatty acids. The fatty acid composition in theoil and the fatty acid composition of the total lipid are generallysimilar; thus, an increase or decrease in the concentration of PUFAs inthe total lipid will correspond with an increase or decrease in theconcentration of PUFAs in the oil, and vice versa.

“Neutral lipids” refer to those lipids commonly found in cells in lipidbodies as storage fats and are so called because at cellular pH, thelipids bear no charged groups. Generally, they are completely non-polarwith no affinity for water. Neutral lipids generally refer to mono-,di-, and/or triesters of glycerol with fatty acids, also calledmonoacylglycerol, diacylglycerol or triacylglycerol, respectively, orcollectively, acylglycerols. A hydrolysis reaction must occur to releasefree fatty acids from acylglycerols.

The term “triacylglycerols” [“TAGs”] refers to neutral lipids composedof three fatty acyl residues esterified to a glycerol molecule. TAGs cancontain LC-PUFAs and saturated fatty acids, as well as shorter chainsaturated and unsaturated fatty acids.

The term “total fatty acids” [“TFAs”] herein refer to the sum of allcellular fatty acids that can be derivitized to fatty acid methyl esters[“FAMEs”] by the base transesterification method (as known in the art)in a given sample, which may be the biomass or oil, for example. Thus,total fatty acids include fatty acids from neutral lipid fractions(including diacylglycerols, monoacylglycerols and TAGs) and from polarlipid fractions (including the PC and the PE fractions), but not freefatty acids.

The term “total lipid content” of cells is a measure of TFAs as apercent of the dry cell weight [“DCW”], although total lipid content canbe approximated as a measure of FAMEs as a percent of the DCW [“FAMEsDCW”]. Thus, total lipid content [“TFAs % DOW”] is equivalent to, e.g.,milligrams of total fatty acids per 100 milligrams of DCW.

The concentration of a fatty acid in the total lipid is expressed hereinas a weight percent of TFAs [“% TFAs”], e.g., milligrams of the givenfatty acid per 100 milligrams of TFAs. Unless otherwise specificallystated in the disclosure herein, reference to the percent of a givenfatty acid with respect to total lipids is equivalent to concentrationof the fatty acid as % TFAs (e.g., % EPA of total lipids is equivalentto EPA % TFAs).

In some cases, it is useful to express the content of a given fattyacid(s) in a cell as its weight percent of the dry cell weight [“%DCW”]. Thus, for example, EPA % DCW would be determined according to thefollowing formula: (EPA % TFAs)*(TFAs % DCW)]/100. The content of agiven fatty acid(s) in a cell as its weight percent of the dry cellweight [“% DOW”] can be approximated, however, as: (EPA % TFAs)*(FAMEs %DCW)]/100.

The terms “lipid profile” and “lipid composition” are interchangeableand refer to the amount of individual fatty acids contained in aparticular lipid fraction, such as in the total lipid or the oil,wherein the amount is expressed as a weight percent of TFAs. The sum ofeach individual fatty acid present in the mixture should be 100.

The term “fatty acids” refers to long chain aliphatic acids (alkanoicacids) of varying chain lengths, from about C₁₂ to C₂₂, although bothlonger and shorter chain-length acids are known. The predominant chainlengths are between C₁₆ and C₂₂. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of carbon [“C”] atoms in the particular fatty acid and Y is thenumber of double bonds. Additional details concerning thedifferentiation between “saturated fatty acids” versus “unsaturatedfatty acids”, “monounsaturated fatty acids” versus “polyunsaturatedfatty acids” [“PUFAs”], and “omega-6 fatty acids” [“ω-6” or “n-6”]versus “omega-3 fatty acids” [“ω-3” or “n-3”] are provided in U.S. Pat.No. 7,238,482, which is hereby incorporated herein by reference.

Nomenclature used to describe PUFAs herein is given in Table 3. In thecolumn titled “Shorthand Notation”, the omega-reference system is usedto indicate the number of carbons, the number of double bonds and theposition of the double bond closest to the omega carbon, counting fromthe omega carbon, which is numbered 1 for this purpose. The remainder ofthe Table summarizes the common names of ω-3 and ω-6 fatty acids andtheir precursors, the abbreviations that will be used throughout thespecification and the chemical name of each compound.

TABLE 3 Nomenclature of Polyunsaturated Fatty Acids And PrecursorsShorthand Common Name Abbreviation Chemical Name Notation Myristic —tetradecanoic 14:0 Palmitic Palmitate hexadecanoic 16:0 Palmitoleic —9-hexadecenoic 16:1 Stearic — octadecanoic 18:0 Oleic —cis-9-octadecenoic 18:1 Linoleic LA cis-9,12-octadecadienoic 18:2 ω-6γ-Linolenic GLA cis-6,9,12-octadecatrienoic 18:3 ω-6 Eicosadienoic EDAcis-11,14-eicosadienoic 20:2 ω-6 Dihomo-γ- DGLAcis-8,11,14-eicosatrienoic 20:3 ω-6 Linolenic Arachidonic ARAcis-5,8,11,14- 20:4 ω-6 eicosatetraenoic α-Linolenic ALA cis-9,12,15-18:3 ω-3 octadecatrienoic Stearidonic STA cis-6,9,12,15- 18:4 ω-3octadecatetraenoic Eicosatrienoic ETrA cis-11,14,17-eicosatrienoic 20:3ω-3 Sciadonic SCI cis-5,11,14-eicosatrienoic 20:3b ω-6 Juniperonic JUPcis-5,11,14,17- 20:4b ω-3 eicosatetraenoic Eicosa- ETA cis-8,11,14,17-20:4 ω-3 tetraenoic eicosatetraenoic Eicosa- EPA cis-5,8,11,14,17- 20:5ω-3 pentaenoic eicosapentaenoic Docosa- DTA cis-7,10,13,16- 22:4 ω-6tetraenoic docosatetraenoic Docosa- DPAn-6 cis-4,7,10,13,16- 22:5 ω-6pentaenoic docosapentaenoic Docosa- DPA cis-7,10,13,16,19- 22:5 ω-3pentaenoic docosapentaenoic Docosa- DHA cis-4,7,10,13,16,19- 22:6 ω-3hexaenoic docosahexaenoic

The term “long-chain polyunsaturated fatty acid” [“LC-PUFA”] refers tothose PUFAs that have chain lengths of C₂₀ or greater. Thus, the termLC-PUFA includes at least EDA, DGLA, ARA, ETrA, ETA, EPA, DTA, DPAn-6,DPA and DHA.

A metabolic pathway, or biosynthetic pathway, in a biochemical sense,can be regarded as a series of chemical reactions occurring in orderwithin a cell, catalyzed by enzymes, to achieve either the formation ofa metabolic product to be used or stored by the cell, or the initiationof another metabolic pathway (then called a flux generating step). Manyof these pathways are elaborate, and involve a step by step modificationof the initial substance to shape it into a product having the exactchemical structure desired.

The term “PUFA biosynthetic pathway” refers to a metabolic process thatconverts oleic acid to ω-6 fatty acids such as LA, EDA, GLA, DGLA, ARA,DRA, DTA and DPAn-6 and ω-3 fatty acids such as ALA, STA, ETrA, ETA,EPA, DPA and DHA. This process is well described in the literature(e.g., see Intl. App. Pub. No. WO 2006/052870). Briefly, this processinvolves elongation of the carbon chain through the addition of carbonatoms and desaturation of the molecule through the addition of doublebonds, via a series of special elongation and desaturation enzymestermed “PUFA biosynthetic pathway enzymes” that are present in theendoplasmic reticulum membrane. More specifically, “PUFA biosyntheticpathway enzymes” refer to any of the following enzymes (and genes whichencode said enzymes) associated with the biosynthesis of a PUFA,including: Δ4 desaturase, Δ5 desaturase, Δ6 desaturase, Δ12 desaturase,Δ15 desaturase, Δ17 desaturase, Δ9 desaturase, Δ8 desaturase, Δ9elongase, C_(14/16) elongase, C_(16/18) elongase, C_(18/20) elongaseand/or C_(20/22) elongase.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a fattyacid or precursor of interest. Despite use of the omega-reference systemthroughout the specification to refer to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system. Ofparticular interest herein are: Δ8 desaturases; Δ5 desaturases; Δ17desaturases; Δ2 desaturases; Δ15 desaturases; Δ9 desaturases; Δ6desaturases; and Δ4 desaturases. Δ17 desaturases, and also Δ5desaturases, are also occasionally referred to as “omega-3 desaturases”,“w-3 desaturases”, and/or “ω-3 desaturases”, based on their ability toconvert ω-6 fatty acids into their ω-3 counterparts.

The term “elongase” refers to a polypeptide that can elongate a fattyacid carbon chain to produce an acid 2 carbons longer than the fattyacid substrate that the elongase acts upon. This process of elongationoccurs in a multi-step mechanism in association with fatty acidsynthase, as described in Intl. App. Pub. No. WO 2005/047480. Examplesof reactions catalyzed by elongase systems are the conversion of GLA toDGLA, STA to ETA, ARA to DTA and EPA to DPA. In general, the substrateselectivity of elongases is somewhat broad but segregated by both chainlength and the degree and type of unsaturation. For example, a C_(14/16)elongase will utilize a C₁₄ substrate (e.g., myristic acid), a C_(16/18)elongase will utilize a C₁₆ substrate (e.g., palmitate), a C_(18/20)elongase will utilize a C₁₈ substrate (e.g., LA, ALA, GLA, STA) and aC_(20/22) elongase (also known as a C20 elongase or Δ5 elongase as theterms can be used interchangeably) will utilize a C₂₀ substrate (e.g.,ARA, EPA). For the purposes herein, two distinct types of C_(18/20)elongases can be defined: a Δ6 elongase will catalyze conversion of GLAand STA to DGLA and ETA, respectively, while a Δ9 elongase is able tocatalyze the conversion of LA and ALA to EDA and ETrA, respectively.

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme, such as adesaturase or elongase, can convert substrate to product. The conversionefficiency is measured according to the following formula:([producty]/[substrate+product])*100, where ‘product’ includes theimmediate product and all products in the pathway derived from it.

The term “C₁₈ to C₂₀ elongation conversion efficiency” refers to theefficiency by which C_(18//20) elongases can convert C₁₈ substrates(i.e., LA, ALA, GLA, STA) to C₂₀ products (i.e., EDA, ETrA, DGLA, ETA).These C_(18//20) elongases can be either Δ9 elongases or Δ6 elongases.

The terms “Δ9 elongation conversion efficiency” and “Δ9 elongaseconversion efficiency” refer to the efficiency by which Δ9 elongase canconvert C₁₈ substrates (i.e., LA, ALA) to C₂₀ products (i.e., EDA,ETrA).

The terms “Δ4 desaturation conversion efficiency” and “Δ4 desaturaseconversion efficiency” refer to the efficiency by which Δ4 desaturasecan convert substrates (i.e., DTA, DPAn-3) to products (i.e., DPAn-6,DHA).

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of oil (Weete, In: Fungal Lipid Biochemistry,2nd Ed., Plenum, 1980). Generally, the cellular oil content ofoleaginous microorganisms follows a sigmoid curve, wherein theconcentration of lipid increases until it reaches a maximum at the latelogarithmic or early stationary growth phase and then graduallydecreases during the late stationary and death phases (Yongmanitchai andWard, Appl. Environ. Microbiol., 57:419-25 (1991)). It is not uncommonfor oleaginous microorganisms to accumulate in excess of about 25% oftheir dry cell weight as oil. Oleaginous microorganisms include variousbacteria, algae, euglenoids, moss, fungi (e.g., Mortierella), yeast andstramenopiles (e.g., Schizochytrium).

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that can make oil. Examples of oleaginous yeast include, but areno means limited to, the following genera: Yarrowia, Candida,Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

The term “fermentable carbon source” means a carbon source that amicroorganism will metabolize to derive energy. Typical carbon sourcesinclude, but are not limited to: monosaccharides, disaccharides,oligosaccharides, polysaccharides, alkanes, fatty acids, esters of fattyacids, glycerol, monoglycerides, diglycerides, triglycerides, carbondioxide, methanol, formaldehyde, formate and carbon-containing amines.

As used herein the term “biomass” refers specifically to spent or usedcellular material from the fermentation of a recombinant production hostproducing PUFAs in commercially significant amounts, wherein thepreferred production host is a recombinant strain of an oleaginous yeastof the genus Yarrowia. The biomass may be in the form of whole cells,whole cell lysates, homogenized cells, partially hydrolyzed cellularmaterial, and/or partially purified cellular material (e.g., microbiallyproduced oil).

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, “nucleic acid fragment” and “isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by a single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deoxycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

As used herein, a nucleic acid fragment is “hybridizable” to anothernucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule,when a single-stranded form of the nucleic acid fragment can anneal tothe other nucleic acid fragment under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), which ishereby incorporated herein by reference, particularly Chapter 11 andTable 11.1.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410(1993)). In general, a sequence of ten or more contiguous amino acids orthirty or more nucleotides is necessary in order to identify putativelya polypeptide or nucleic acid sequence as homologous to a known proteinor gene. Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation, such as in situ hybridization ofbacterial colonies or bacteriophage plaques. In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to specifically identify and/or isolatea nucleic acid fragment comprising the sequence.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine.

As used herein, the terms “homology” and “homologous” are usedinterchangeably. They refer to nucleic acid fragments wherein changes inone or more nucleotide bases do not affect the ability of the nucleicacid fragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragmentssuch as deletion or insertion of one or more nucleotides that do notsubstantially alter the functional properties of the resulting nucleicacid fragment relative to the initial, unmodified fragment.

Moreover, the skilled artisan recognizes that homologous nucleic acidsequences are also defined by their ability to hybridize, undermoderately stringent conditions, e.g., 0.5×SSC, 0.1% SDS, 60° C., withthe sequences exemplified herein, or to any portion of the nucleotidesequences disclosed herein and which are functionally equivalentthereto. Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes, Part I, Chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,New York (1993); and Current Protocols in Molecular Biology, Chapter 2,Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York(1995).

As used herein, the term “percent identity” refers to a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. “Identity” alsomeans the degree of sequence relatedness between polypeptide orpolynucleotide sequences, as the case may be, as determined by thepercentage of match between compared sequences. “Percent identity” and“percent similarity” can be readily calculated by known methods,including but not limited to those described in: 1) ComputationalMolecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and, 5) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Preferred methods to determine percent identity are designed to give thebest match between the sequences tested. Methods to determine percentidentity and percent similarity are codified in publicly availablecomputer programs. Sequence alignments and percent identity calculationsmay be performed using the MegAlign™ program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences is performed using the “Clustal method ofalignment” which encompasses several varieties of the algorithmincluding the “Clustal V method of alignment” and the “Clustal W methodof alignment” (described by Higgins and Sharp, CABIOS, 5:151-153 (1989);Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) andfound in the MegAlign™ (version 8.0.2) program of the LASERGENEbioinformatics computing suite (DNASTAR Inc.). Default parameters formultiple protein alignment using the Clustal W method of alignmentcorrespond to GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay DivergentSeqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=GonnetSeries, DNA Weight Matrix=IUB with the ‘slow-accurate’ option. Afteralignment of the sequences using either Clustal program, it is possibleto obtain a “percent identity” by viewing the “sequence distances” tablein the program.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include any integerpercentage from 34% to 100%, such as 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99%. Also, of interest is any full-length or partial complementof this isolated nucleotide fragment. Suitable nucleic acid fragmentsnot only have the above homologies but typically encode a polypeptidehaving at least 50 amino acids, preferably at least 100 amino acids,more preferably at least 150 amino acids, still more preferably at least200 amino acids, and most preferably at least 250 amino acids.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These oligonucleotide building blocks are annealed and thenligated to form gene segments that are then enzymatically assembled toconstruct the entire gene. Accordingly, the genes can be tailored foroptimal gene expression based on optimization of nucleotide sequence toreflect the codon bias of the host cell. The skilled artisan appreciatesthe likelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cell,where sequence information is available. For example, the codon usageprofile for Yarrowia lipolytica is provided in U.S. Pat. No. 7,125,672.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, and which may refer to the coding region alone or may includeregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene that is introduced intothe host organism by gene transfer. Foreign genes can comprise nativegenes inserted into a non-native organism, native genes introduced intoa new location within the native host, or chimeric genes. A “transgene”is a gene that has been introduced into the genome by a transformationprocedure. A “codon-optimized gene” is a gene having its frequency ofcodon usage designed to mimic the frequency of preferred codon usage ofthe host cell.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, enhancers, silencers, 5′ untranslated leader sequence (e.g.,between the transcription start site and the translation initiationcodon), introns, polyadenylation recognition sequences, RNA processingsites, effector binding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The terms “3′ non-coding sequence” and “transcription terminator” referto DNA sequences located downstream of a coding sequence. This includespolyadenylation recognition sequences and other sequences encodingregulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The 3′ region can influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” or “mRNA” refersto the RNA that is without introns and which can be translated intoprotein by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to, and derived from, mRNA.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence. That is, the coding sequence is under thetranscriptional control of the promoter. Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA. Expression mayalso refer to translation of mRNA into a polypeptide.

“Transformation” refers to the transfer of a nucleic acid molecule intoa host organism. The nucleic acid molecule may be a plasmid thatreplicates autonomously, for example, or, it may integrate into thegenome of the host organism. Host organisms containing the transformednucleic acid fragments are referred to as “transgenic” or “recombinant”or “transformed” or “transformant” organisms.

“Stable transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance (i.e.,the nucleic acid fragment is “stably integrated”). In contrast,“transient transformation” refers to the transfer of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance.

The terms “plasmid” and “vector” refer to an extra chromosomal elementoften carrying genes that are not part of the central metabolism of thecell, and usually in the form of circular double-stranded DNA fragments.Such elements may be autonomously replicating sequences, genomeintegrating sequences, phage or nucleotide sequences, linear orcircular, of a single- or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction that is capable of introducing anexpression cassette(s) into a cell.

The term “expression cassette” refers to a fragment of DNA comprisingthe coding sequence of a selected gene and regulatory sequencespreceding (5′ non-coding sequences) and following (3′ non-codingsequences) the coding sequence that are required for expression of theselected gene product. Thus, an expression cassette is typicallycomposed of: 1) a promoter sequence; 2) a coding sequence [“ORF”]; and,3) a 3′ untranslated region (i.e., a terminator) that, in eukaryotes,usually contains a polyadenylation site. The expression cassette(s) isusually included within a vector, to facilitate cloning andtransformation. Different expression cassettes can be transformed intodifferent organisms including bacteria, yeast, plants and mammaliancells, as long as the correct regulatory sequences are used for eachhost.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); 3) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and, 5) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

As previously described, genes encoding LPLATs are found in alleukaryotic cells, based on their intimate role in de novo synthesis andremodeling of glycerophospholipids, wherein LPLATs remove acyl-CoA fattyacids from the cellular acyl-CoA pool and acylate variouslysophospholipid substrates at the sn-2 position in the phospholipidpool. Publicly available sequences encoding LPLATs include ScAle1 (SEQID NO:9), ScLPAAT (SEQ ID NO:18), MaLPAAT1 (SEQ ID NO:15) and CeLPCAT(SEQ ID NO:2). The ScAle1 (SEQ ID NO:9) and ScLPAAT (SEQ ID NO:18)protein sequences were used as a query to identify orthologs from thepublic Y. lipolytica protein database (the “Yeast project Genolevures”(Center for Bioinformatics, LaBR1, Talence Cedex, France) (see alsoDujon, B. et al., Nature, 430(6995):35-44 (2004)). Based on analysis ofthe best hits, the Ale1 and LPAAT orthologs from Yarrowia lipolytica areidentified herein as YlAle1 (SEQ ID NO:11) and YlLPAAT1 (SEQ ID NO:17),respectively (see Example 5, infra).

When the sequence of a particular LPLAT gene or protein within apreferred host organism is not known, the LPLAT sequences set forthherein as SEQ ID NOs:2, 9, 11, 15, 17 and 18, or portions of them, maybe used to search for LPLAT homologs in the same or other algal, fungal,oomycete, euglenoid, stramenopiles, yeast or plant species usingsequence analysis software. In general, such computer software matchessimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications. Use of softwarealgorithms, such as the BLASTP method of alignment with a low complexityfilter and the following parameters: Expect value=10, matrix=Blosum 62(Altschul, et al., Nucleic Acids Res., 25:3389-3402 (1997)), iswell-known for comparing any LPLAT protein against a database of nucleicor protein sequences and thereby identifying similar known sequenceswithin a preferred host organism.

Use of a software algorithm to comb through databases of known sequencesis particularly suitable for the isolation of homologs having arelatively low percent identity to publicly available LPLAT sequences,such as those described in SEQ ID NOs:2, 9, 11, 15, 17 and 18. It ispredictable that isolation would be relatively easier for LPLAT homologsof at least about 70%-85% identity to publicly available LPLATsequences. Further, those sequences that are at least about 85%-90%identical would be particularly suitable for isolation and thosesequences that are at least about 90%-95% identical would be the mostfacilely isolated.

LPLAT homologs can also be identified by the use of motifs unique to theLPLAT enzymes. These motifs likely represent regions of the LPLATprotein that are important to the structure, stability or activity ofthe protein and these motifs are useful as diagnostic tools for therapid identification of novel LPLAT genes.

A variety of LPLAT motifs have been proposed, with slight variationbased on the specific species that are included in analyzed alignments.For example, Shindou et al. (Biochem. Biophys. Res. Comm., 383:320-325(2009)) proposed the following membrane bound O-acyltransferase[“MBOAT”] family motifs to be important for LPLAT activity, based onalignment of sequences from Homo sapiens, Gallus gallus, Danio rerio andCaenorhabditis elegans: WD, WHGxxxGYxxxF (SEQ ID NO:23), YxxxxF (SEQ IDNO:24) and YxxxYFxxH (SEQ ID NO:25). Of these, the WD, WHGxxxGYxxxF andYxxxxF motifs are present in ScAle and YlAle1, but the YxxxYFxxH motifis not. Alternate non-plant motifs for Ale1 homologs are also describedin U.S. Pat. Pub. No. 2008-0145867-A1; specifically, these include:M-[V/I]-[L/I]-xxK-[L/V/I]-xxxxxxDG (SEQ ID NO:26),RxKYYxxWxxx-[E/D]-[A/G]xxxxGxG-[F/Y]-xG (SEQ ID NO:27),EX₁₁WNX₂-[T/V]-X₂W (SEQ ID NO:28) and SAxWHGxxPGYxx-[T/F]-F (SEQ IDNO:29).

Similarly, Lewin, T. W. et al. (Biochemistry, 38:5764-5771 (1999)) andYamashita et al. (Biochim, Biophys. Acta, 1771:1202-1215 (2007))proposed the following 1-acyl-sn-glycerol-3-phosphate acyltransferase[“LPAAT”] family motifs to be important for LPLAT activity, based onalignment of sequences from bacteria, yeast, nematodes and mammals:NHxxxxD (SEQ ID NO:19), GxxFI-[D/R]-R (SEQ ID NO:30), EGTR (SEQ IDNO:20) and either [V/I]-[P/X]-[I/V/L]-[I/V]-P-[V/I] (SEQ ID NO:31) orIVPIVM (SEQ ID NO:32). The NHxxxxD and EGTR motifs are present inMaLPAAT1, YlLPAAT1 and CeLPCAT, but the other motifs are not.

Based on publicly available Ale1, LPCAT and LPAAT protein sequences,including those described herein, the instant invention concerns thefollowing MBOAT family motifs: M(V/I)LxxKL (SEQ ID NO:3), RxKYYxxW (SEQID NO:4), SAxWHG (SEQ ID NO:5) and EX₁₁WNX₂-[T/V]-X₂W (SEQ ID NO:28).Similarly, 1-acyl-sn-glycerol-3-phosphate acyltransferase family motifsare those set forth as: NHxxxxD (SEQ ID NO:19) and EGTR (SEQ ID NO:20).

Alternatively, publicly available LPLAT sequences or their motifs may behybridization reagents for the identification of homologs. Hybridizationmethods are well known to those of ordinary skill in the art as notedabove.

Any of the LPLAT nucleic acid fragments or any identified homologs maybe used to isolate genes encoding homologous proteins from the same orother algal, fungal, oomycete, euglenoid, stramenopiles, yeast or plantspecies. Isolation of homologous genes using sequence-dependentprotocols is well known in the art. Examples of sequence-dependentprotocols include, but are not limited to: 1) methods of nucleic acidhybridization; 2) methods of DNA and RNA amplification, as exemplifiedby various uses of nucleic acid amplification technologies, such aspolymerase chain reaction [“PCR”] (U.S. Pat. No. 4,683,202); ligasechain reaction [“LCR”](Tabor, S. et al., Proc. Natl. Acad. Sci. U.S.A.,82:1074 (1985)); or strand displacement amplification [“SDA”] (Walker,et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)); and, 3) methodsof library construction and screening by complementation.

For example, genes encoding proteins or polypeptides similar to publiclyavailable LPLAT genes or their motifs could be isolated directly byusing all or a portion of those publicly available nucleic acidfragments as DNA hybridization probes to screen libraries from anydesired organism using well known methods. Specific oligonucleotideprobes based upon the publicly available nucleic acid sequences can bedesigned and synthesized by methods known in the art (Maniatis, supra).Moreover, the entire sequences can be used directly to synthesize DNAprobes by methods known to the skilled artisan, such as random primersDNA labeling, nick translation or end-labeling techniques, or RNA probesusing available in vitro transcription systems. In addition, specificprimers can be designed and used to amplify a part or the full length ofthe publicly available sequences or their motifs. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full-length DNA fragments under conditions of appropriatestringency.

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown (Thein and Wallace, “The use of oligonucleotides as specifichybridization probes in the Diagnosis of Genetic Disorders”, in HumanGenetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp33-50, IRL: Herndon, Va.; Rychlik, W., In Methods in Molecular Biology,White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: CurrentMethods and Applications. Humania: Totowa, N.J.).

Generally two short segments of available LPLAT sequences may be used inPCR protocols to amplify longer nucleic acid fragments encodinghomologous genes from DNA or RNA. PCR may also be performed on a libraryof cloned nucleic acid fragments wherein the sequence of one primer isderived from the available nucleic acid fragments or their motifs. Thesequence of the other primer takes advantage of the presence of thepolyadenylic acid tracts to the 3′ end of the mRNA precursor encodinggenes.

Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al., Proc. Natl. Acad. Sci. U.S.A.,85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of theregion between a single point in the transcript and the 3′ or 5′ end.Primers oriented in the 3′ and 5′ directions can be designed from theavailable sequences. Using commercially available 3′ RACE or 5′ RACEsystems (e.g., BRL, Gaithersburg, Md.), specific 3′ or 5′ cDNA fragmentscan be isolated (Ohara et al., Proc. Natl. Acad. Sci. U.S.A., 86:5673(1989); Loh et al., Science, 243:217 (1989)).

Based on any of these well-known methods just discussed, it would bepossible to identify and/or isolate LPLAT gene homologs in any preferredeukaryotic organism of choice. The activity of any putative LPLAT genecan readily be confirmed by expression of the gene within aLC-PUFA-producing host organism, since the C₁₈ to C₂₀ elongation and/orΔ4 desaturation are increased relative to those within an organismlacking the LPLAT transgene (supra).

It has been previously hypothesized that LPCATs could be important inthe accumulation of EPA in the TAG fraction of Yarrowia lipolytica (U.S.Pat. Pub. No. 2006-0115881-A1). As described therein, this hypothesiswas based on the following studies: 1) Stymne S, and A. K. Stobart(Biochem J., 223(2):305-314 (1984)), who hypothesized that the exchangebetween the acyl-CoA pool and PC pool may be attributed to the forwardand backward reaction of LPCAT; 2) Domergue, F. et al. (J. Bio. Chem.,278:35115 (2003)), who suggested that accumulation of GLA at the sn-2position of PC and the inability to efficiently synthesize ARA in yeastwas a result of the elongation step involved in PUFA biosynthesisoccurring within the acyl-CoA pool, while Δ5 and Δ6 desaturation stepsoccurred predominantly at the sn-2 position of PC; 3) Abbadi, A. et al.(The Plant Cell, 16:2734-2748 (2004)), who suggested that LPCAT plays acriticial role in the successful reconstitution of a Δ6 desaturase/Δ6elongase pathway, based on analysis on the constraints of PUFAaccumulation in transgenic oilseed plants; and, 4) Intl. App. Pub. No.WO 2004/076617 A2 (Renz, A. et al.), who provided a gene encoding LPCATfrom Caenorhabditis elegans (T06E8.1) [“CeLPCAT”] that substantiallyimproved the efficiency of elongation in a genetically introduced Δ6desaturase/Δ6 elongase pathway in S. cerevisiae fed with exogenous fattyacid substrates suitable for Δ6 elongation. Renz et al. concluded thatLPCAT allowed efficient and continuous exchange of the newly synthesizedfatty acids between phospholipids and the acyl-CoA pool, sincedesaturases catalyze the introduction of double bonds in PC-coupledfatty acids while elongases exclusively catalyze the elongation of CoAesterified fatty acids (acyl-CoAs). However, Intl. App. Pub. No. WO2004/076617 did not teach the effect of CeLPCAT on Δ6 elongationconversion efficiency in host cells that were not exogenously fed fattyacids, Δ5 elongation conversion efficiency, or Δ4 desaturationconversion efficiency.

Herein, it is demonstrated that LPAAT and LPCAT are indeed important inthe accumulation of EPA and DHA in the TAG fraction of Yarrowialipolytica. However, unexpectedly, it was found that over-expression ofLPLATs can result in an improvement in the Δ9 elongase conversionefficiency and/or Δ4 desaturase conversion efficiency. As previouslydefined, conversion efficiency is a term that refers to the efficiencyby which a particular enzyme, such as a Δ4 desaturase or Δ9 elongase,can convert substrate to product. Thus, in a strain engineered toproduce EPA, improvement in Δ9 elongase conversion efficiency wasdemonstrated to result in increased EPA % TFAs or EPA % DCW. Similarly,improvement in Δ9 elongase and/or Δ4 desaturase conversion efficiency ina strain engineered to produce DHA was demonstrated to result inincreased DHA TFAs or DHA % DCW.

PUFA desaturations occur on phospholipids, while fatty acid elongationsoccur on acyl-CoAs. Based on previous studies, it was therefore expectedthat LPLAT over-expression would result in improved desaturations due toimproved substrate availability in phospholipids, while expression ofLPLATs was not expected to result in improved elongations that requireimproved substrate availability in the CoA pool.

Despite these assumptions, Example 5 demonstrates that LPLAT expressiondid not improve the conversion efficiency of all desaturations instrains of Yarrowia producing DHA, in a comparable manner. Specifically,the conversion efficiency of Δ4 desaturase was selectively improved,while similar improvements were not found in Δ2, Δ8, Δ5 or Δ17desaturations. It is hypothesized that Δ4 desaturase was thereforelimiting as a result of limited availability of the DPA substrate inphospholipids.

Additionally, Examples 4 and 5 demonstrate that LPLAT expression, basedon at least one stably integrated polynucleotide encoding the LPLATpolypeptide, significantly improved the Δ9 elongase conversionefficiency in strains of Yarrowia producing EPA and DHA, respectively.Surprisingly, however, the LPLATs did not also result in a comparableimprovement in the efficiency of the C_(20/22) elongation of EPA to DPAin DHA strains. Generally, there was no significant change in the totallipid content in strains over-expressing LPLATs versus those that werenot.

Clearly, broad generalizations are difficult concerning the effect ofLPLAT over-expression in host cells producing PUFAs. Instead, the effectof LPLAT activity must be considered based on subsets of desaturases andelongases having specific activity (i.e., Δ2 desaturase, Δ8 desaturase,Δ5 desaturase, Δ17 desaturase, Δ4 desaturase, Δ9 elongase, C_(14/16)elongase, C_(16/18) elongase, C_(18/20) elongase [“also Δ6 elongase”],C_(20/22) elongase [“also Δ5 elongase”]).

On the basis of the above discussion, in one embodiment herein, methodsfor improving C₁₈ to C₂₀ elongation conversion efficiency in aLC-PUFA-producing recombinant oleaginous microbial host cell areprovided, wherein said method comprises:

a) introducing into said LC-PUFA-producing recombinant host cell atleast one isolated polynucleotide encoding a polypeptide having at leastacyl-CoA:lysophospholipid acyltransferase activity wherein thepolypeptide is selected from the group consisting of:

-   -   (i) a polypeptide having at least 45% amino acid identity, based        on the Clustal W method of alignment, when compared to an amino        acid sequence selected from the group consisting of SEQ ID NO:9        (ScAle1) and SEQ ID NO:11 (YlAle1);    -   (ii) a polypeptide having at least one membrane bound        O-acyltransferase protein family motif selected from the group        consisting of: M(V/I)LxxKL (SEQ ID NO:3), RxKYYxxW (SEQ ID        NO:4), SAxWHG (SEQ ID NO:5) and EX₁₁WNX₂-[T/V]-X₂W (SEQ ID        NO:28);    -   (iii) a polypeptide having at least 90% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence as set forth in SEQ ID NO:2 (CeLPCAT);    -   (iv) a polypeptide having at least 43.9% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence selected from the group consisting of SEQ ID        NO:15 (MaLPAAT1), SEQ ID NO:17 (YlLPAAT1) and SEQ ID NO:18        (ScLPAAT1); and,    -   (v) a polypeptide having at least one        1-acyl-sn-glycerol-3-phosphate acyltransferase protein family        motif selected from the group consisting of: NHxxxxD (SEQ ID        NO:19) and EGTR (SEQ ID NO:20);

wherein the at least one isolated polynucleotide encoding a polypeptidehaving at least acyl-CoA:lysophospholipid acyltransferase activity isoperably linked to at least one regulatory sequence, said regulatorysequence being the same or different; and,

b) growing the oleaginous microbial host cell;

wherein the C₁₈ to C₂₀ elongation conversion efficiency of theoleaginous microbial host cell is increased relative to the control hostcell.

In preferred embodiments, the increase in C₁₈ to C₂₀ elongationconversion efficiency is at least 4% in at least one LC-PUFA-producingoleaginous microbial host cell, based on at least one stably integratedpolynucleotide encoding the LPLAT polypeptide, when compared to thecontrol host cell, although any increase in C₁₈ to C₂₀ elongationconversion efficiency greater than 4% is especially preferred, includingincreases of at least about 4-10%, more preferred at least about 10-20%,more preferred at least about 20-40%, and most preferred at least about40-60% or greater.

For example, in one method demonstrated herein, the increase in C₁₈ toC₂₀ elongation conversion efficiency is at least 13% in an EPA-producinghost cell when compared to the control host cell and the increase in C₁₈to C₂₀ elongation conversion efficiency is at least 4% in aDHA-producing host cell when compared to the control host cell.

Similarly, methods are also described herein for increasing Δ4desaturation conversion efficiency in a LC-PUFA-producing oleaginousmicrobial recombinant host cell, wherein said method comprises:

a) introducing into said LC-PUFA-producing recombinant host cell atleast one isolated polynucleotide encoding a polypeptide having at leastacyl-CoA:lysophospholipid acyltransferase activity wherein thepolypeptide is selected from the group consisting of:

-   -   (i) a polypeptide having at least 45% amino acid identity, based        on the Clustal W method of alignment, when compared to an amino        acid sequence selected from the group consisting of SEQ ID NO:9        (ScAle1) and SEQ ID NO:11 (YlAle1);    -   (ii) a polypeptide having at least one membrane bound        O-acyltransferase protein family motif selected from the group        consisting of: M(V/I)LxxKL (SEQ ID NO:3), RxKYYxxW (SEQ ID        NO:4), SAxWHG (SEQ ID NO:5) and EX₁₁WNX₂-[T/V]-X₂W (SEQ ID        NO:28);    -   (iii) a polypeptide having at least 90% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence as set forth in SEQ ID NO:2 (CeLPCAT);    -   (iv) a polypeptide having at least 43.9% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence selected from the group consisting of SEQ ID        NO:15 (MaLPAAT1), SEQ ID NO:17 (YlLPAAT1) and SEQ ID NO:18        (ScLPAAT1); and,    -   (v) a polypeptide having at least one        1-acyl-sn-glycerol-3-phosphate acyltransferase protein family        motif selected from the group consisting of: NHxxxxD (SEQ ID        NO:19) and EGTR (SEQ ID NO:20);

wherein the at least one isolated polynucleotide encoding a polypeptidehaving at least acyl-CoA:lysophospholipid acyltransferase activity isoperably linked to at least one regulatory sequence, said regulatorysequence being the same or different; and,

b) growing the oleaginous microbial host cell;

wherein the Δ4 desaturation conversion efficiency of the oleaginousmicrobial host cell is increased relative to the control host cell.

In preferred embodiments, the increase in Δ4 desaturation conversionefficiency is at least 5% in at least one LC-PUFA-producing oleaginousmicrobial host cell, based on at least one stably integratedpolynucleotide encoding the LPLAT polypeptide, when compared to thecontrol host cell, although any increase in Δ4 desaturation conversionefficiency greater than 5% is especially preferred, including increasesof at least about 5-10%, more preferred at least about 10-20%, morepreferred at least about 20-40%, and most preferred at least about40-60% or greater.

For example, in one method demonstrated herein, the increase in Δ4desaturation conversion efficiency in a DHA-producing host was at least18% when compared to the control host cell.

Recombinant host cells are also described herein, in addition to themethods set forth above. Specifically, these recombinant host cellscomprise at least one isolated polynucleotide encoding a polypeptidehaving at least acyl-CoA:lysophospholipid acyltransferase activity,wherein the polypeptide is selected from the group consisting of:

-   -   (a) a polypeptide having at least 45% amino acid identity, based        on the Clustal W method of alignment, when compared to an amino        acid sequence selected from the group consisting of SEQ ID NO:9        (ScAle1) and SEQ ID NO:11 (YlAle1);    -   (b) a polypeptide having at least one membrane bound        O-acyltransferase protein family motif selected from the group        consisting of: M(V/I)LxxKL (SEQ ID NO:3), RxKYYxxW (SEQ ID        NO:4), SAxWHG (SEQ ID NO:5) and EX₁₁WNX₂-[T/V]-X₂W (SEQ ID        NO:28);    -   (c) a polypeptide having at least 90% amino acid identity, based        on the Clustal W method of alignment, when compared to an amino        acid sequence as set forth in SEQ ID NO:2 (CeLPCAT);    -   (d) a polypeptide having at least 43.9% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence selected from the group consisting of SEQ ID        NO:15 (MaLPAAT1), SEQ ID NO:17 (YlLPAAT1) and SEQ ID NO:18        (ScLPAAT1); and,    -   (e) a polypeptide having at least one        1-acyl-sn-glycerol-3-phosphate acyltransferase family motif        selected from the group consisting of: NHxxxxD (SEQ ID NO:19)        and EGTR (SEQ ID NO:20);

wherein the at least one isolated polynucleotide encoding a polypeptidehaving at least acyl-CoA:lysophospholipid acyltransferase activity isoperably linked to at least one regulatory sequence, said regulatorysequence being the same or different, and the recombinant host cellsfurther have at least one improvement selected from the group consistingof:

a) an increase in C₁₈ to C₂₀ elongation conversion efficiency in atleast one LC PUFA-producing oleaginous microbial host cell when comparedto the control host cell;

b) an increase in Δ4 desaturation conversion efficiency in at least oneLC PUFA-producing oleaginous microbial host cell when compared to thecontrol host cell.

In preferred host cells, the polynucleotide encoding the polypeptidehaving at least acyl-CoA:lysophospholipid acyltransferase activity isstably integrated; and, further wherein the host cell has at least oneimprovement selected from the group consisting of:

a) an increase in C₁₈ to C₂₀ elongation conversion efficiency of atleast 4% in at least one long-chain polyunsaturated fatty acid-producingoleaginous microbial host cell when compared to a control host cell;and,

b) an increase in Δ4 desaturation conversion efficiency of at least 5%in at least one long-chain polyunsaturated fatty acid-producingoleaginous microbial host cell when compared to a control host cell.

In more preferred host cells, having at least one stably integratedpolynucleotide encoding the LPLAT polypeptide, the at least oneimprovement is selected from the group consisting of:

a) an increase in C₁₈ to C₂₀ elongation conversion efficiency of atleast 13% in an EPA-producing host cell when compared to the controlhost cell;

b) an increase of at least 9% EPA of TFAs in an EPA-producing host cellwhen compared to the control host cell;

c) an increase in C₁₈ to C₂₀ elongation conversion efficiency of atleast of at least 4% in a DHA-producing host cell when compared to thecontrol host cell;

d) an increase of at least 2% EPA of TFAs in a DHA-producing host cellwhen compared to the control host cell;

e) an increase in Δ4 desaturation conversion efficiency of at least 18%in a DHA-producing host cell when compared to the control host cell;and,

f) an increase of at least 9% DHA of TFAs in a DHA-producing host cellwhen compared to the control host cell.

Of course, one of skill in the art should understand that theimprovements described above should be considered as exemplary, but notlimiting to the invention herein.

Based on the above improvements, one of skill in the art will appreciatethe value of expressing a LPLAT in a recombinant host cell that isproducing long-chain PUFAs, such EDA, DGLA, ARA, DTA, DPAn-6, ETrA, ETA,EPA, DPA and DHA, if it is desirable to optimize the production of thesefatty acids.

Standard resource materials that are useful to make recombinantconstructs describe, inter alia: 1) specific conditions and proceduresfor construction, manipulation and isolation of macromolecules, such asDNA molecules, plasmids, etc.; 2) generation of recombinant DNAfragments and recombinant expression constructs; and, 3) screening andisolation of clones. See, Sambrook, J., Fritsch, E. F. and Maniatis, T.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); bySilhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with GeneFusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984);and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken,N.J. (1987).

In general, the choice of sequences included in the construct depends onthe desired expression products, the nature of the host cell and theproposed means of separating transformed cells versus non-transformedcells. The skilled artisan is aware of the genetic elements that must bepresent on the plasmid vector to successfully transform, select andpropagate host cells containing the chimeric gene. Typically, however,the vector or cassette contains sequences directing transcription andtranslation of the relevant gene(s), a selectable marker and sequencesallowing autonomous replication or chromosomal integration. Suitablevectors comprise a region 5′ of the gene that controls transcriptionalinitiation, i.e., a promoter, the gene coding sequence, and a region 3′of the DNA fragment that controls transcriptional termination, i.e., aterminator. It is most preferred when both control regions are derivedfrom genes from the transformed host cell, although they need not bederived from genes native to the production host.

Transcription initiation regions or promoters useful for drivingexpression of heterologous genes or portions of them in the desired hostcell are numerous and well known. These control regions may comprise apromoter, enhancer, silencer, intron sequences, 3′ UTR and/or 5′ UTRregions, and protein and/or RNA stabilizing elements. Such elements mayvary in their strength and specificity. Virtually any promoter, i.e.,native, synthetic, or chimeric, capable of directing expression of thesegenes in the selected host cell is suitable, although transcriptionaland translational regions from the host species are particularly useful.Expression in a host cell can occur in an induced or constitutivefashion. Induced expression occurs by inducing the activity of aregulatable promoter operably linked to the LPLAT gene of interest,while constitutive expression occurs by the use of a constituitivepromoter.

3′ non-coding sequences encoding transcription termination regions maybe provided in a recombinant construct and may be from the 3′ region ofthe gene from which the initiation region was obtained or from adifferent gene. A large number of termination regions are known andfunction satisfactorily in a variety of hosts when utilized in both thesame and different genera and species from which they were derived.Termination regions may also be derived from various genes native to thepreferred hosts. The termination region is usually selected more forconvenience rather than for any particular property.

Particularly useful termination regions for use in yeast are derivedfrom a yeast gene, particularly Saccharomyces, Schizosaccharomyces,Candida, Yarrowia or Kluyveromyces. The 3′-regions of mammalian genesencoding γ-interferon and α-2 interferon are also known to function inyeast. The 3′-region can also be synthetic, as one of skill in the artcan utilize available information to design and synthesize a 3′-regionsequence that functions as a transcription terminator. A terminationregion may be unnecessary, but is highly preferred.

The vector may also comprise a selectable and/or scorable marker, inaddition to the regulatory elements described above. Preferably, themarker gene is an antibiotic resistance gene such that treating cellswith the antibiotic results in growth inhibition, or death, ofuntransformed cells and uninhibited growth of transformed cells. Forselection of yeast transformants, any marker that functions in yeast isuseful with resistance to kanamycin, hygromycin and the amino glycosideG418 and the ability to grow on media lacking uracil, lysine, histine orleucine being particularly useful.

Merely inserting a gene (e.g., encoding a LPLAT) into a cloning vectordoes not ensure its expression at the desired rate, concentration,amount, etc. In response to the need for a high expression rate, manyspecialized expression vectors have been created by manipulating anumber of different genetic elements that control transcription, RNAstability, translation, protein stability and location, oxygenlimitation, and secretion from the host cell. Some of the manipulatedfeatures include: the nature of the relevant transcriptional promoterand terminator sequences, the number of copies of the cloned gene andwhether the gene is plasmid-borne or integrated into the genome of thehost cell, the final cellular location of the synthesized protein, theefficiency of translation and correct folding of the protein in the hostorganism, the intrinsic stability of the mRNA and protein of the clonedgene within the host cell and the codon usage within the cloned gene,such that its frequency approaches the frequency of preferred codonusage of the host cell. Each of these may be used in the methods andhost cells described herein to further optimize expression of LPLATgenes.

For example, LPLAT expression can be increased at the transcriptionallevel through the use of a stronger promoter (either regulated orconstitutive) to cause increased expression, by removing/deletingdestabilizing sequences from either the mRNA or the encoded protein, orby adding stabilizing sequences to the mRNA (U.S. Pat. No. 4,910,141).Alternately, additional copies of the LPLAT genes may be introduced intothe recombinant host cells to thereby increase EPA and/or DHA productionand accumulation, either by cloning additional copies of genes within asingle expression construct or by introducing additional copies into thehost cell by increasing the plasmid copy number or by multipleintegration of the cloned gene into the genome.

After a recombinant construct is created comprising at least onechimeric gene comprising a promoter, a LPLAT open reading frame [“ORF”]and a terminator, it is placed in a plasmid vector capable of autonomousreplication in the host cell or is directly integrated into the genomeof the host cell. Integration of expression cassettes can occur randomlywithin the host genome or can be targeted through the use of constructscontaining regions of homology with the host genome sufficient to targetrecombination with the host locus. Where constructs are targeted to anendogenous locus, all or some of the transcriptional and translationalregulatory regions can be provided by the endogenous locus.

When two or more genes are expressed from separate replicating vectors,each vector may have a different means of selection and should lackhomology to the other construct(s) to maintain stable expression andprevent reassortment of elements among constructs. Judicious choice ofregulatory regions, selection means and method of propagation of theintroduced construct(s) can be experimentally determined so that allintroduced genes are expressed at the necessary levels to provide forsynthesis of the desired products.

Constructs comprising the gene(s) of interest may be introduced into ahost cell by any standard technique. These techniques includetransformation, e.g., lithium acetate transformation (Methods inEnzymology, 194:186-187 (1991)), biolistic impact, electroporation,microinjection, vacuum filtration or any other method that introducesthe gene of interest into the host cell.

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence, for example, in an expression cassette, isreferred to herein as “transformed” or “recombinant” or “transformant”.The transformed host will have at least one copy of the expressionconstruct and may have two or more, depending upon whether the gene isintegrated into the genome, amplified, or is present on anextrachromosomal element having multiple copy numbers.

The transformed host cell can be identified by selection for a markercontained on the introduced construct. Alternatively, a separate markerconstruct may be co-transformed with the desired construct, as manytransformation techniques introduce many DNA molecules into host cells.

Typically, transformed hosts are selected for their ability to grow onselective media, which may incorporate an antibiotic or lack a factornecessary for growth of the untransformed host, such as a nutrient orgrowth factor. An introduced marker gene may confer antibioticresistance, or encode an essential growth factor or enzyme, therebypermitting growth on selective media when expressed in the transformedhost. Selection of a transformed host can also occur when the expressedmarker protein can be detected, either directly or indirectly.Additional selection techniques are described in U.S. Pat. No. 7,238,482and U.S. Pat. No. 7,259,255.

Regardless of the selected host or expression construct, multipletransformants must be screened to obtain a strain displaying the desiredexpression level and pattern. For example, Juretzek et al. (Yeast,18:97-113 (2001)) note that the stability of an integrated DNA fragmentin Yarrowia lipolytica is dependent on the individual transformants, therecipient strain and the targeting platform used. Such screening may beaccomplished by Southern analysis of DNA blots (Southern, J. Mol. Biol.,98:503 (1975)), Northern analysis of mRNA expression (Kroczek, J.Chromatogr. Biomed. Appl., 618(1-2):133-145 (1993)), Western analysis ofprotein expression, phenotypic analysis or GC analysis of the PUFAproducts.

The metabolic process wherein oleic acid is converted to LC-PUFAsinvolves elongation of the carbon chain through the addition of carbonatoms and desaturation of the molecule through the addition of doublebonds. This requires a series of special desaturation and elongationenzymes present in the endoplasmic reticulum membrane. However, as seenin FIGS. 1A and B and as described below, multiple alternate pathwaysexist for LC-PUFA production.

Specifically, FIGS. 1A and B depict the pathways described below. Allpathways require the initial conversion of oleic acid to linoleic acid[“LA”], the first of the ω-6 fatty acids, by a Δ2 desaturase. Then,using the “Δ9 elongase/Δ8 desaturase pathway” and LA as substrate,long-chain ω-6 fatty acids are formed as follows: 1) LA is converted toeicosadienoic acid [“EDA”] by a Δ9 elongase; 2) EDA is converted todihomo-γ-linolenic acid [“DGLA”] by a Δ8 desaturase; 3) DGLA isconverted to arachidonic acid [“ARA”] by a Δ5 desaturase; 4) ARA isconverted to docosatetraenoic acid [“DTA”] by a C_(20/22) elongase; and,5) DTA is converted to docosapentaenoic acid [“DPAn-6”] by a Δ4desaturase.

The “Δ9 elongase/Δ8 desaturase pathway” can also use α-linolenic acid[“ALA”] as substrate to produce long-chain ω-3 fatty acids asfollows: 1) LA is converted to ALA, the first of the ω-3 fatty acids, bya Δ15 desaturase; 2) ALA is converted to eicosatrienoic acid [“ETrA”] bya Δ9 elongase; 3) ETrA is converted to eicosatetraenoic acid [“ETA”] bya Δ8 desaturase; 4) ETA is converted to eicosapentaenoic acid [“EPA”] bya Δ5 desaturase; 5) EPA is converted to docosapentaenoic acid [“DPA”] bya C_(20/22) elongase; and, 6) DPA is converted to docosahexaenoic acid[“DHA”] by a Δ4 desaturase. Optionally, ω-6 fatty acids may be convertedto ω-3 fatty acids. For example, ETA and EPA are produced from DGLA andARA, respectively, by Δ17 desaturase activity. Advantageously for thepurposes herein, the Δ9 elongase/Δ8 desaturase pathway enablesproduction of an EPA oil that lacks significant amounts of γ-linolenicacid [“GLA”].

Alternate pathways for the biosynthesis of ω-3/ω-6 fatty acids utilize aΔ6 desaturase and C_(18/20) elongase, that is, the “Δ6 desaturase/Δ6elongase pathway”. More specifically, LA and ALA may be converted to toGLA and stearidonic acid [“STA”], respectively, by a Δ6 desaturase;then, a C_(18/20) elongase converts GLA to DGLA and/or STA to ETA.

A LC-PUFA-producing recombinant host cell will possess at least one ofthe biosynthetic pathways described above, whether this pathway isnative to the host cell or is genetically engineered. Preferably, thehost cell will be capable of producing at least about 2-5% LC-PUFAs inthe total lipids of the recombinant host cell, more preferably at leastabout 5-15% LC-PUFAs in the total lipids, more preferably at least about15-35% LC-PUFAs in the total lipids, more preferably at least about35-50% LC-PUFAs in the total lipids, more preferably at least about50-65% LC-PUFAs in the total lipids and most preferably at least about65-75% LC-PUFAs in the total lipids. The structural form of the LC-PUFAsis not limiting; thus, for example, the EPA or DHA may exist in thetotal lipids as free fatty acids or in esterified forms such asacylglycerols, phospholipids, sulfolipids or glycolipids.

A variety of eukaryotic microbial organisms, including bacteria, yeast,algae, stramenopile, oomycete, euglenoid and/or fungus, can produce (orcan be engineered to produce) LC-PUFAs. These may include hosts thatgrow on a variety of feedstocks, including simple or complexcarbohydrates, fatty acids, organic acids, oils, glycerols and alcohols,and/or hydrocarbons over a wide range of temperature and pH values.

Preferred microbial hosts are oleaginous organisms. These oleaginousorganisms are naturally capable of oil synthesis and accumulation,wherein the total oil content can comprise greater than about 25% of thedry cell weight, more preferably greater than about 30% of the dry cellweight, and most preferably greater than about 40% of the dry cellweight. Various bacteria, algae, euglenoids, moss, fungi, yeast andstramenopiles are naturally classified as oleaginous. Within this broadgroup of hosts, of particular interest are those organisms thatnaturally produce ω-3/ω-6 fatty acids. For example, ARA, EPA and/or DHAis produced via Cyclotella sp., Crypthecodinium sp., Mortierella sp.,Nitzschia sp., Pythium, Thraustochytrium sp. and Schizochytrium sp.Thus, for example, transformation of Mortierella alpina, which iscommercially used for production of ARA, with any of the present LPLATgenes under the control of inducible or regulated promoters could yielda transformant organism capable of synthesizing increased quantities ofARA. The method of transformation of M. alpina is described by Mackenzieet al. (Appl. Environ. Microbiol., 66:4655 (2000)). Similarly, methodsfor transformation of Thraustochytriales microorganisms (e.g.,Thraustochytrium, Schizochytrium) are disclosed in U.S. Pat. No.7,001,772. In alternate embodiments, a non-oleaginous organism can begenetically modified to become oleaginous, e.g., yeast such asSaccharomyces cerevisiae (U.S. Pat. Pub. No. 2007/0015237-A1).

In more preferred embodiments, the microbial host cells are oleaginousyeast. Genera typically identified as oleaginous yeast include, but arenot limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces. More specifically,illustrative oil-synthesizing yeast include: Rhodosporidium toruloides,Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C.tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorulaglutinus, R. graminis and Yarrowia lipolytica (formerly classified asCandida lipolytica). Most preferred is the oleaginous yeast Yarrowialipolytica; and, in a further embodiment, most preferred are the Y.lipolytica strains designated as ATCC #76982, ATCC #20362, ATCC #8862,ATCC #18944 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G.,Bioresour. Technol., 82(1):43-9 (2002)).

Specific teachings applicable for engineering ARA, EPA and DHAproduction in Y. lipolytica are provided in U.S. Pat. Pub. No.2006-0094092-A1, U.S. Pat. Pub. No. 2006-0115881-A1, U.S. Pat. Pub. No.2009-0093543-A1 and U.S. Pat. Pub. No. 2006-0110806-A1, respectively.These references also describe the preferred method of expressing genesin Yarrowia lipolytica by integration of a linear DNA fragment into thegenome of the host, preferred promoters, termination regions,integration loci and disruptions, and preferred selection methods whenusing this particular host species.

One of skill in the art would be able to use the cited teachings in U.S.Pat. Pub. No. 2006-0094092-A1, U.S. Pat. Pub. No. 2006-0115881-A1, U.S.Pat. Pub. No. 2009-0093543-A1 and U.S. Pat. Pub. No. 2006-0110806-A1 torecombinantly engineer other host cells for PUFA production.

The transformed recombinant host cell is grown under conditions thatoptimize expression of chimeric genes (e.g., encoding desaturases,elongases, LPLATs, etc.) and produce the greatest and the mosteconomical yield of LC-PUFA(s). In general, media conditions may beoptimized by modifying the type and amount of carbon source, the typeand amount of nitrogen source, the carbon-to-nitrogen ratio, the amountof different mineral ions, the oxygen level, growth temperature, pH,length of the biomass production phase, length of the oil accumulationphase and the time and method of cell harvest.

Yarrowia lipolytica are generally grown in a complex media such as yeastextract-peptone-dextrose broth [“YPD”] or a defined minimal media thatlacks a component necessary for growth and thereby forces selection ofthe desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Fermentation media for the methods and host cells described herein mustcontain a suitable carbon source, such as are taught in U.S. Pat. No.7,238,482 and U.S. patent application Ser. No. 12/641,929 (filed Dec.19, 2009). Although it is contemplated that the source of carbonutilized may encompass a wide variety of carbon-containing sources,preferred carbon sources are sugars, glycerol and/or fatty acids. Mostpreferred is glucose, sucrose, invert sucrose, fructose and/or fattyacids containing between 10-22 carbons. For example, the fermentablecarbon source can be selected from the group consisting of invertsucrose, glucose, fructose and combinations of these, provided thatglucose is used in combination with invert sucrose and/or fructose.

The term “invert sucrose”, also referred to herein as “invert sugar”,refers to a mixture comprising equal parts of fructose and glucoseresulting from the hydrolysis of sucrose. Invert sucrose may be amixture comprising 25 to 50% glucose and 25 to 50% fructose. Invertsucrose may also comprise sucrose, the amount of which depends on thedegree of hydrolysis.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic(e.g., urea or glutamate) source. In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins and other components knownto those skilled in the art suitable for the growth of the high EPA-and/or DHA-producing host cells and the promotion of the enzymaticpathways for EPA and/or DHA production. Particular attention is given toseveral metal ions, such as Fe⁺², Cu⁺², Mn⁺², Co+², Zn⁺² and Mg⁺², thatpromote synthesis of lipids and PUFAs (Nakahara, T. et al., Ind. Appl.Single Cell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).

Preferred growth media for the methods and host cells described hereinare common commercially prepared media, such as Yeast Nitrogen Base(DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growthmedia may also be used and the appropriate medium for growth of Yarrowialipolytica will be known by one skilled in the art of microbiology orfermentation science. A suitable pH range for the fermentation istypically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 ispreferred as the range for the initial growth conditions. Thefermentation may be conducted under aerobic or anaerobic conditions,wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of EPA and/or DHA in Yarrowia lipolytica. This approach isdescribed in U.S. Pat. No. 7,238,482, as are various suitablefermentation process designs (i.e., batch, fed-batch and continuous) andconsiderations during growth.

In some aspects, the primary product is oleaginous microbial biomass. Assuch, isolation and purification of the LC-PUFA-containing oils from thebiomass may not be necessary (i.e., wherein the whole cell biomass isthe product).

However, certain end uses and/or product forms may require partialand/or complete isolation/purification of the LC-PUFA-containing oilfrom the biomass, to result in partially purified biomass, purified oil,and/or purified LC-PUFAs. Fatty acids, including PUFAs, may be found inthe host microorganisms as free fatty acids or in esterified forms suchas acylglycerols, phospholipids, sulfolipids or glycolipids. These fattyacids may be extracted from the host cells through a variety of meanswell-known in the art. One review of extraction techniques, qualityanalysis and acceptability standards for yeast lipids is that of Z.Jacobs (Critical Reviews in Biotechnology, 12(5/6):463-491 (1992)). Abrief review of downstream processing is also available by A. Singh andO. Ward (Adv. Appl. Microbiol., 45:271-312 (1997)).

In general, means for the purification of fatty acids (includingLC-PUFAs) may include extraction (e.g., U.S. Pat. No. 6,797,303 and U.S.Pat. No. 5,648,564) with organic solvents, sonication, supercriticalfluid extraction (e.g., using carbon dioxide), saponification andphysical means such as presses, or combinations thereof. See U.S. Pat.No. 7,238,482.

Many food and feed products incorporate ω-3 and/or ω-6 fatty acids,particularly ALA, GLA, ARA, EPA, DPA and DHA. It is contemplated thatoleaginous yeast biomass comprising LC-PUFAs, partially purified biomasscomprising LC-PUFAs, purified oil comprising LC-PUFAs, and/or purifiedLC-PUFAs made by the methods and host cells described herein impart thehealth benefits, upon ingestion of foods or feed improved by theiraddition. These oils can be added to food analogs, drinks, meatproducts, cereal products, baked foods, snack foods and dairy products,to name a few. See U.S. Pat. Appl. Pub. No. 2006-0094092.

These compositions may impart health benefits by being added to medicalfoods including medical nutritionals, dietary supplements, infantformula and pharmaceuticals. The skilled artisan will appreciate theamount of the oils to be added to food, feed, dietary supplements,nutriceuticals, pharmaceuticals, and other ingestible products as toimpart health benefits. Health benefits from ingestion of these oils aredescribed in the art, known to the skilled artisan and continuouslyinvestigated. Such an amount is referred to herein as an “effective”amount and depends on, among other things, the nature of the ingestedproducts containing these oils and the physical conditions they areintended to address.

EXAMPLES

The present invention is further described in the following Examples,which illustrate reductions to practice of the invention but do notcompletely define all of its possible variations.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by: 1) Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(Maniatis); 2) T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions; Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); and, 3) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience, Hoboken, N.J. (1987).

Materials and methods suitable for the maintenance and growth ofmicrobial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, 2nd ed., Sinauer Associates Sunderland, Mass.(1989). All reagents, restriction enzymes and materials used for thegrowth and maintenance of microbial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), NewEngland Biolabs, Inc. (Beverly, Mass.), GIBCO/BRL (Gaithersburg, Md.),or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.E. coli strains were typically grown at 37° C. on Luria Bertani [“LB”]plates.

General molecular cloning was performed according to standard methods(Sambrook et al., supra). DNA sequence was generated on an ABI Automaticsequencer using dye terminator technology (U.S. Pat. No. 5,366,860; EP272,007) using a combination of vector and insert-specific primers.Sequence editing was performed in Sequencher (Gene Codes Corporation,Ann Arbor, Mich.). All sequences represent coverage at least two timesin both directions.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “pmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s), “kB” means kilobase(s), “DCW” means dry cell weight, and “TFAs”means total fatty acids.

Nomenclature for Expression Cassettes

The structure of an expression cassette will be represented by a simplenotation system of “X::Y::Z”, wherein X describes the promoter fragment,Y describes the gene fragment, and Z describes the terminator fragment,which are all operably linked to one another.

Transformation and Cultivation of Yarrowia lipolytica

Yarrowia lipolytica strain ATCC #20362 was purchased from the AmericanType Culture Collection (Rockville, Md.). Yarrowia lipolytica strainswere routinely grown at 28-30° C. in several media (e.g., YPD agarmedium, Basic Minimal Media [“MM”], Minimal Media+Uracil [“MMU”],Minimal Media+Leucine+Lysine [“MMLeuLys”], Minimal Media+5-FluorooroticAcid [“MM+5-FOA”], High Glucose Media [“HGM”] and Fermentation medium[“FM”]), as described in U.S. Pat. Appl. Pub. No. 2009-0093543-A1.

Transformation of Y. lipolytica was performed as described in U.S. Pat.Appl. Pub. No. 2009-0093543-A1.

Fatty Acid Analysis of Yarrowia lipolytica

For fatty acid [“FA”] analysis, cells were collected by centrifugationand lipids were extracted as described in Bligh, E. G. & Dyer, W. J.(Can. J. Biochem. Physiol., 37:911-917 (1959)). Fatty acid methyl esters[“FAMEs”] were prepared by transesterification of the lipid extract withsodium methoxide (Roughan, G., and Nishida I., Arch Biochem Biophys.,276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia cells (0.5 mL culture)were harvested, washed once in distilled water, and dried under vacuumin a Speed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) and a knownamount of C15:0 triacylglycerol (C15:0 TAG; Cat. No. T-145, Nu-CheckPrep, Elysian, Minn.) was added to the sample, and then the sample wasvortexed and rocked for 30 min at 50° C. After adding 3 drops of 1 MNaCl and 400 μl hexane, the sample was vortexed and spun. The upperlayer was removed and analyzed by GC.

FAME peaks recorded via GC analysis were identified by their retentiontimes, when compared to that of known fatty acids, and quantitated bycomparing the FAME peak areas with that of the internal standard (C15:0TAG) of known amount. Thus, the approximate amount (μg) of any fattyacid FAME [“μg FAME”] is calculated according to the formula: (area ofthe FAME peak for the specified fatty acid/area of the standard FAMEpeak)*(μg of the standard C15:0 TAG), while the amount (μg) of any fattyacid [“μg FA”] is calculated according to the formula: (area of the FAMEpeak for the specified fatty acid/area of the standard FAME peak)*(μg ofthe standard C15:0 TAG)*0.9503, since 1 μg of C15:0 TAG is equal to0.9503 μg fatty acids. Note that the 0.9503 conversion factor is anapproximation of the value determined for most fatty acids, which rangebetween 0.95 and 0.96.

The lipid profile, summarizing the amount of each individual fatty acidas a weight percent of TFAs, was determined by dividing the individualFAME peak area by the sum of all FAME peak areas and multiplying by 100.

For quantitating the amount of an individual fatty acid or the totalfatty acids as a weight percent of the dry cell weight [“% DCW”], cellsfrom 10 mL of the culture were collected by centrifugation, washed oncewith 10 mL water and collected by centrifugation again. Cells wereresuspended in 1-2 mL water, poured into a pre-weighed aluminiumweighing pan, and rinsed with 1-2 mL water that was also added to thesame weighing pan. The pan was placed under vacuum at 80° C. overnight.The pan was weighed and the DCW calculated by subtracting the weight ofthe empty pan. Determination of the fatty acid as a % DCW can then becalculated based on either μg FAME or μg FA as a fraction of the μg DCW(for example, FAME % DCW was calculated as μg FAME/μg DCW*100).

Example 1 Generation of Yarrowia lipolytica Strain Y8406 to Produceabout 51% EPA of Total Fatty Acids

The present Example describes the construction of strain Y8406, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing about 51% EPArelative to the total lipids via expression of a Δ9 elongase/Δ8desaturase pathway. This strain was used as the EPA-producing host cellin Example 4.

The development of strain Y8406 (FIG. 2) required the construction ofstrains Y2224, Y4001, Y4001U, Y4036, Y4036U, L135, L135U9, Y8002,Y8006U6, Y8069, Y8069U, Y8154, Y8154U, Y8269 and Y8269U.

Generation of Y4036U Strain

Briefly, strain Y8406 was derived from Yarrowia lipolytica ATCC #20362via construction of strain Y2224 (a FOA resistant mutant from anautonomous mutation of the Ura3 gene of wildtype Yarrowia strain ATCC#20362), strain Y4001 (producing 17% EDA with a Leu− phenotype), strainY4001U1 (Leu− and Ura−), strain Y4036 (producing 18% DGLA with a Leu−phenotype) and strain Y4036U (Leu− and Ura−). Further details regardingthe construction of strains Y2224, Y4001, Y4001U, Y4036 and Y4036U aredescribed in the General Methods of U.S. Pat. App. Pub. No.2008-0254191, hereby incorporated herein by reference.

The final genotype of strain Y4036U with respect to wild type Yarrowialipolytica ATCC #20362 was Ura3−, YAT1::ME3S::Pex16, EXP1::EgD9eS::Lip1,FBAINm::EgD9eS::Lip2, GPAT::EgD9e::Lip2, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, GPD::FmD12::Pex20, YAT1::FmD12::OCT (wherein FmD12is a Fusarium moniliforme Δ2 desaturase gene [U.S. Pat. No. 7,504,259];MESS is a codon-optimized C_(16/18) elongase gene, derived fromMortierella alpina [U.S. Pat. No. 7,470,532]; EgD9e is a Euglenagracilis Δ9 elongase gene [U.S. Pat. No. 7,645,604]; EgD9eS is acodon-optimized Δ9 elongase gene, derived from Euglena gracilis [U.S.Pat. No. 7,645,604]; EgD8M is a synthetic mutant Δ8 desaturase [U.S.Pat. No. 7,709,239], derived from Euglena gracilis [U.S. Pat. No.7,256,033]).

Generation of L135 Strain (Ura3+, Leu−, Δpex3) with Chromosomal DeletionOf Pex3

Construction of strain L135 is described in Example 12 of Intl. App.Pub. No. WO 2009/046248, hereby incorporated herein by reference.Briefly, construct pY157 was used to knock out the chromosomal geneencoding the peroxisome biogenesis factor 3 protein [peroxisomalassembly protein Peroxin 3 or “Pex3p”] in strain Y4036U, therebyproducing strain L135 (also referred to as strain Y4036 (Δpex3)).Knockout of the chromosomal Pex3 gene in strain L135, as compared to instrain Y4036 (whose native Pex3p had not been knocked out) resulted inthe following: higher lipid content (TFAs DCW) (ca. 6.0% versus 4.7%),higher DGLA % TFAs (46% versus 19%), higher DGLA % DCW (ca. 2.8% versus0.9%) and reduced LA % TFAs (12% versus 30%). Additionally, the Δ9elongase percent conversion efficiency was increased from ca. 48% instrain Y4036 to 83% in strain L135.

The final genotype of strain L135 with respect to wildtype Yarrowialipolytica ATCC #20362 was Ura3+, Leu−, Pex3−, unknown1−,YAT1::ME3S::Pex16, EXP1::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2,GPAT::EgD9e::Lip2, FBAINm::EgD8M::Pex20, EXP1::EgD8M::Pex16,GPD::FmD12::Pex20, YAT1::FmD12::OCT.

Generation of L135U9 (Leu−, Ura3−) Strain

Strain L135U was created via temporary expression of the Cre recombinaseenzyme in plasmid pY116 (FIG. 3; SEQ ID NO:33; described in Example 7 ofIntl. App. Pub. No. WO 2008/073367, hereby incorporated herein byreference) within strain L135 to produce a Leu− and Ura− phenotype.Plasmid pY116 was used for transformation of freshly grown L135 cellsaccording to the General Methods. The transformant cells were platedonto MMLeuUra plates and maintained at 30° C. for 3 to 4 days. Threecolonies were picked, inoculated into 3 mL liquid YPD media at 30° C.and shaken at 250 rpm/min for 1 day. The cultures were diluted to1:50,000 with liquid MMLeuUra media, and 100 μL was plated onto new YPDplates and maintained at 30° C. for 2 days. Eight colonies were pickedfrom each of three plates (24 colonies total) and streaked onto MMLeuand MMLeuUra selection plates. The colonies that could grow on MMLeuUraplates but not on MMLeu plates were selected and analyzed by GC toconfirm the presence of C20:2 (EDA). One strain, having a Leu− and Ura−phenotype, was designated as L135U9.

Generation of Y8002 Strain to Produce about 32% ARA of TFAs

Construct pZKSL-5S5A5 (FIG. 4A; SEQ ID NO:34) was generated to integratethree Δ5 desaturase genes into the Lys loci of strain L135U9, to therebyenable production of ARA. The pZKSL-5S5A5 plasmid contained thefollowing components:

TABLE 4 Description of Plasmid pZKSL-5S5A5 (SEQ ID NO: 34) RE Sites AndNucleotides Within SEQ ID NO: 34 Description Of Fragment And ChimericGene Components AscI/BsiWI 720 bp 5′ portion of Yarrowia Lys5 gene(GenBank Accession (5925-6645) No. M34929; labeled as “lys5 5′ region”in Figure) PacI/SphI 689 bp 3′ portion of Yarrowia Lys5 gene (GenBankAccession (2536-3225) No. M34929; labeled as “Lys5-3′” in Figure)EcoRI/BsiWI FBAIN::EgD5SM::Pex20, comprising: (9338-6645) FBAIN:Yarrowia lipolytica FBAIN promoter (U.S. Pat. No. 7,202,356); EgD5SM:Synthetic mutant Δ5 desaturase (SEQ ID NO: 35; U.S. patent Pub. No.2010-0075386-A1), derived from Euglena gracilis (U.S. patent No.7,678,560) (labeled as “ED5S” in Figure); Pex20: Pex20 terminatorsequence from Yarrowia Pex20 gene (GenBank Accession No. AF054613)PmeI/ClaI YAT1::EaD5SM::OCT, comprising: (11503-1) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; U.S. patentapplication Pub. No. 2006-0094102-A1); EaD5SM: Synthetic, mutant Δ5desaturase (SEQ ID NO: 37; U.S. patent Pub. No. 2010-0075386-A1),derived from Euglena anabaena (U.S. patent application Pub. No.2008-0274521-A1) (labeled as “EaD5S” in Figure); OCT: OCT terminatorsequence of Yarrowia OCT gene (GenBank Accession No. X69988) ClaI/PacIEXP1::EgD5M::Pex16, comprising: (1-2536) EXP1: Yarrowia lipolyticaexport protein (EXP1) promoter (labeled as “EXP” in Figure; Intl. App.Pub. No. WO 2006/052870); EgD5M: Mutant Δ5 desaturase (SEQ ID NO: 90;U.S. patent Pub. No. 2010-0075386-A1) with elimination of internalEcoRI, BglII, HindIII and NcoI restriction enzyme sites, derived fromEuglena gracilis (U.S. Pat. No. 7,678,560) (labeled as “Euglena D5WT” inFigure); Pex16: Pex16 terminator sequence from Yarrowia Pex16 gene(GenBank Accession No. U75433) EcoRI/PmeI Yarrowia Leu2 gene (GenBankAccession No. M37309) (9360-11503)

The pZKSL-5S5A5 plasmid was digested with AscI/SphI, and then used fortransformation of strain L135U9 according to the General Methods. Thetransformant cells were plated onto MMUraLys plates and maintained at30° C. for 2 to 3 days. Single colonies were then re-streaked ontoMMUraLys plates, and then inoculated into liquid MMUraLys at 30° C. andshaken at 250 rpm/min for 2 days. The cells were subjected to fatty acidanalysis, according to the General Methods.

GC analyses showed the presence of ARA in the transformants containingthe 3 chimeric genes of pZKSL-5S5A5, but not in the parent L135U9strain. Five strains (i.e., #28, #62, #73, #84 and #95) that producedabout 32.2%, 32.9%, 34.4%, 32.1% and 38.6% ARA of TFAs were designatedas strains Y8000, Y8001, Y8002, Y8003 and Y8004, respectively. Furtheranalyses showed that the three chimeric genes of pZKSL-5S5A5 were notintegrated into the Lys5 site in the Y8000, Y8001, Y8002, Y8003 andY8004 strains. All strains possessed a Lys+ phenotype.

The final genotype of strains Y8000, Y8001, Y8002, Y8003 and Y8004 withrespect to wildtype Yarrowia lipolytica ATCC #20362 was Ura−, Pex3−unknown 1−, unknown 2−, Leu+, Lys+, YAT1::ME3S::Pex16,GPD::FmD12::Pex20, YAT1::FmD12::Oct, GPAT::EgD9e::Lip2,FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD5SM::Pex20, EXP1::EgD5M::Pex16,YAT1::EaD5SM::Oct.

Generation of Y8006 Strains to Produce about 41% ARA of TFAs

Construct pZP3-Pa777U (FIG. 4B; SEQ ID NO:39; described in Table 9 ofU.S. Pat. Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein byreference) was generated to integrate three Δ17 desaturase genes intothe Pox3 loci (GenBank Accession No. AJ001301) of strain Y8002.

The pZP3-Pa777U plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8002 according to the General Methods. Thetransformant cells were plated onto MM plates and maintained at 30° C.for 2 to 3 days. Single colonies were then re-streaked onto MM plates,and then inoculated into liquid MM at 30° C. and shaken at 250 rpm/minfor 2 days. The cells were subjected to fatty acid analysis, accordingto the General Methods.

GC analyses showed the presence of 26% to 31% EPA of TFAs in most of theselected 96 transformants containing the 3 chimeric genes ofpZP3-Pa777U, but not in the parent Y8002 strain. Strain #69 producedabout 38% EPA of TFAs and was designated as Y8007. There was one strain(i.e., strain #9) that did not produce EPA, but produced about 41% ARAof TFAs. This strain was designated as Y8006. Based on the lack of EPAproduction in strain Y8006, its genotype with respect to wildtypeYarrowia lipolytica ATCC #20362 is assumed to be Pex3−, unknown 1−,unknown 2−, unknown 3−, Leu+, Lys+, Ura+, YAT1::ME3S::Pex16,GPD::FmD12::Pex20, YAT1::FmD12::Oct, GPAT::EgD9e::Lip2,FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD5SM::Pex20, EXP1::EgD5M::Pex16,YAT1::EaD5SM::Oct.

In contrast, the final genotype of strain Y8007 with respect to wildtypeYarrowia lipolytica ATCC #20362 was Pex3−, unknown 1−, unknown 2−,unknown 3−, Leu+, Lys+, Ura+, YAT1::ME3S::Pex16, GPD::FmD12::Pex20,YAT1::FmD12::Oct, GPAT::EgD9e::Lip2, FBAINm::EgD9eS::Lip2,EXP1::EgD9eS::Lip1, FBAINm::EgD8M::Pex20, EXP1::EgD8M::Pex16,FBAIN::EgD5SM::Pex20, EXP1::EgD5M::Pex16, YAT1::EaD5SM::Oct,YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16, FBAINm::PaD17::Aco (whereinPaD17 is a Pythium aphanidermatum Δ17 desaturase [U.S. Pat. No.7,556,949] and PaD17S is a codon-optimized Δ17 desaturase, derived fromPythium aphanidermatum [U.S. Pat. No. 7,556,949].

Integration of the 3 chimeric genes of pZP3-Pa777U into the Pox3 loci(GenBank Accession No. AJ001301) in strains Y8006 and Y8007 was notconfirmed.

Generation of Strain Y8006U6 (Ura3−)

To disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ ID NO:40;described in Table 15 of U.S. Pat. Appl. Pub. No. 2009-0093543-A1,hereby incorporated herein by reference) was used to integrate a Ura3mutant gene into the Ura3 gene of strain Y8006.

Plasmid pZKUM was digested with SalI/Pact, and then used to transformstrain Y8006 according to the General Methods. Following transformation,cells were plated onto MM+5-FOA selection plates and maintained at 30°C. for 2 to 3 days.

A total of 8 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. All 8strains had a Ura− phenotype (i.e., cells could grow on MM+5-FOA plates,but not on MM plates). The cells were scraped from the MM+5-FOA platesand subjected to fatty acid analysis, according to the General Methods.

GC analyses showed the presence of 22.9%, 25.5%, 23.6% 21.6%, 21.6% and25% ARA of TFAs in the pZKUM-transformant strains #1, #2, #4, #5, #6 and#7, respectively, grown on MM+5-FOA plates. These six strains weredesignated as strains Y8006U1, Y8006U2, Y8006U3, Y8006U4, Y8006U5 andY8006U6, respectively (collectively, Y8006U).

Generation of Y8069 Strain to Produce about 37.5% EPA of TFAs

Construct pZP3-Pa777U (FIG. 4B; SEQ ID NO:39; described in Table 9 ofU.S. Pat. Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein byreference) was used to integrate three Δ17 desaturase genes into thePox3 loci (GenBank Accession No. AJ001301) of strain Y8006U6.

The pZP3-Pa777U plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8006U6 according to the General Methods. Thetransformant cells were plated onto MM plates and maintained at 30° C.for 2 to 3 days. Single colonies were then re-streaked onto MM plates,and then inoculated into liquid MM at 30° C. and shaken at 250 rpm/minfor 2 days. The cells were subjected to fatty acid analysis, accordingto the General Methods.

GC analyses showed the presence of EPA in the transformants containingthe 3 chimeric genes of pZP3-Pa777U, but not in the parent Y8006U6strain. Most of the selected 24 strains produced 24-37% EPA of TFAs.Four strains (i.e., #1, #6, #11 and #14) that produced 37.5%, 43.7%,37.9% and 37.5% EPA of TFAs were designated as Y8066, Y8067, Y8068 andY8069, respectively. Integration of the 3 chimeric genes of pZP3-Pa777Uinto the Pox3 loci (GenBank Accession No. AJ001301) of strains Y8066,Y8067, Y8068 and Y8069 was not confirmed.

The final genotype of strains Y8066, Y8067, Y8068 and Y8069 with respectto wildtype Yarrowia lipolytica ATCC #20362 was Ura+, Pex3−, unknown 1−,unknown 2−, unknown 3−, unknown 4−, Leu+, Lys+, YAT1::ME3S::Pex16,GPD::FmD12::Pex20, YAT1::FmD12::Oct, GPAT::EgD9e::Lip2,FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD5SM::Pex20, EXP1::EgD5M::Pex16,YAT1::EaD5SM::Oct, YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16,FBAINm::PaD17::Aco.

Generation of Strain Y8069U (Ura3−)

To disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ ID NO:40;described in Table 15 of U.S. Pat. Appl. Pub. No. 2009-0093543-A1) wasused to integrate a Ura3 mutant gene into the Ura3 gene of strain Y8069,in a manner similar to that described for pZKUM transformation of strainY8006 (supra). A total of 3 transformants were grown and identified topossess a Ura− phenotype.

GC analyses showed the presence of 22.4%, 21.9% and 21.5% EPA of TFAs inthe pZKUM-transformant strains #1, #2 and #3, respectively, grown onMM+5-FOA plates. These three strains were designated as strains Y8069U1,Y8069U2, and Y8069U3, respectively (collectively, Y8069U).

Generation of Strain Y8154 to Produce about 44.8% EPA of TFAs

Construct pZKL2-5 mB89C (FIG. 5B; SEQ ID NO:41) was generated tointegrate one Δ5 desaturase gene, one Δ9 elongase gene, one Δ8desaturase gene, and one Yarrowia lipolytica diacylglycerolcholinephosphotransferase gene (CPT1) into the Lip2 loci (GenBankAccession No. AJ012632) of strain Y8069U3 to thereby enable higher levelproduction of EPA. The pZKL2-5 mB89C plasmid contained the followingcomponents:

TABLE 5 Description of Plasmid pZKL2-5mB89C (SEQ ID NO: 41) RE Sites AndNucleotides Within SEQ ID NO: 41 Description Of Fragment And ChimericGene Components AscI/BsiWI 722 bp 5′ portion of Yarrowia Lip2 gene(labeled as “Lip2.5N” in (730-1) Figure; GenBank Accession No. AJ012632)PacI/SphI 697 bp 3′ portion of Yarrowia Lip2 gene (labeled as “Lip2.3N”in (4141-3438) Figure; GenBank Accession No. AJ012632) SwaI/BsiWIYAT1::YlCPT1::Aco, comprising: (13561-1) YAT1: Yarrowia lipolytica YAT1promoter (labeled as “YAT” in Figure; U.S. patent application Pub. No.2006-0094102-A1); YlCPT1: Yarrowia lipolytica diacylglycerolcholinephosphotransferase gene (SEQ ID NO: 42) (labeled as “Y.lipolytica CPT1 cDNA” in Figure; Intl. App. Pub. No. WO 2006/052870);Aco: Aco terminator sequence from Yarrowia Aco gene (GenBank AccessionNo. AJ001300) PmeI/SwaI FBAIN::EgD8M::Lip1 comprising: (10924-13561)FBAIN: Yarrowia lipolytica FBAIN promoter (U.S. Pat. No. 7,202,356);EgD8M: Synthetic mutant Δ8 desaturase (SEQ ID NO: 44; U.S. Pat. No.7,709,239), derived from Euglena gracilis (“EgD8S”; U.S. Pat. No.7,256,033) (labeled as “D8S-23” in Figure); Lip1: Lip1 terminatorsequence from Yarrowia Lip1 gene (GenBank Accession No. Z50020)PmeI/ClaI YAT1::EgD9eS::Lip2, comprising: (10924-9068) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; U.S. patentapplication Pub. No. 2006-0094102-A1); EgD9eS: codon-optimized Δ9elongase (SEQ ID NO: 46), derived from Euglena gracilis (U.S. Pat. No.7,645,604); Lip2: Lip2 terminator sequence from Yarrowia Lip2 gene(GenBank Accession No. AJ012632) ClaI/EcoRI Yarrowia Ura3 gene (GenBankAccession No. AJ306421) (9068-6999) EcoRI/PacI GPDIN::EgD5SM::ACO,comprising: (6999-4141) GPDIN: Yarrowia lipolytica GPDIN promoter (U.S.Pat. No. 7,459,546); EgD5SM: Synthetic mutant Δ5 desaturase (SEQ ID NO:35; U.S. patent Pub. No. 2010-0075386-A1), derived from Euglena gracilis(U.S. Pat. No. 7,678,560) (labeled as “EgD5S-HPGS” in Figure); Aco: Acoterminator sequence from Yarrowia Aco gene (GenBank Accession No.AJ001300)

The pZKL2-5 mB89C plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8069U3 according to the General Methods. Thetransformant cells were plated onto MM plates and maintained at 30° C.for 3 to 4 days. Single colonies were re-streaked onto MM plates, andthen inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, resuspended in HGM andthen shaken at 250 rpm/min for 5 days. The cells were subjected to fattyacid analysis, according to the General Methods.

GC analyses showed that most of the selected 96 strains producedapproximately 38-44% EPA of TFAs. Seven strains (i.e., #1, #39, #49,#62, #70, #85 and #92) that produced about 44.7%, 45.2%, 45.4%, 44.8%,46.1%, 48.6% and 45.9% EPA of TFAs were designated as strains Y8151,Y8152, Y8153, Y8154, Y8155, Y8156 and Y8157, respectively. Knockout ofthe Lip2 gene was not confirmed in these EPA strains.

The final genotype of strains Y8151, Y8152, Y8153, Y8154, Y8155, Y8156and Y8157 with respect to wildtype Yarrowia lipolytica ATCC #20362 wasUra+, Pex3−, unknown 1−, unknown 2−, unknown 3−, unknown 4−, unknown 5−,Leu+, Lys+, YAT1::ME3S::Pex16, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,GPAT::EgD9e::Lip2, YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1, GPD::FmD12::Pex20,YAT1::FmD12::Oct, EXP1::EgD5M::Pex16, YAT1::EaD5SM::Oct,FBAIN::EgD5SM::Pex20, GPDIN::EgD5SM::Aco, FBAINm::PaD17::Aco,EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1, YAT1::YlCPT::Aco.

Generation of Strain Y8154U1 (Ura3−)

To disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ ID NO:40;described in Table 15 of U.S. Pat. Appl. Pub. No. 2009-0093543-A1) wasused to integrate a Ura3 mutant gene into the Ura3 gene of strain Y8154,in a manner similar to that described for pZKUM transformation of strainY8006 (supra). A total of 8 transformants were grown and identified topossess a Ura− phenotype.

GC analyses showed that there was 23.1% EPA of TFAs in thepZKUM-transformant strain #7. This strain was designated as strainY8154U1.

Generation of Strain Y8269 to Produce about 45.3% EPA of TFAs

Construct pZKL1-2SR9G85 (FIG. 6A; SEQ ID NO:48) was generated tointegrate one DGLA synthase, one Δ12 desaturase gene and one Δ5desaturase gene into the Lip1 loci (GenBank Accession No. Z50020) ofstrain Y8154U1 to thereby enable higher level production of EPA. A DGLAsynthase is a multizyme comprising a Δ9 elongase linked to a Δ8desaturase (U.S. Pat. Appl. Pub. No. 2008-0254191-A1).

The pZKL1-2SR9G85 plasmid contained the following components:

TABLE 6 Description of Plasmid pZKL1-2SR9G85 (SEQ ID NO: 48) RE SitesAnd Nucleotides Within SEQ ID NO: 48 Description Of Fragment AndChimeric Gene Components AscI/BsiWI 809 by 5′ portion of Yarrowia Lip1gene (labeled as “Lip1-5′N” in (4189-3373) Figure; GenBank Accession No.Z50020) PacI/SphI 763 by 3′ portion of Yarrowia Lip1 gene (labeled as“Lip1.3N” in (7666-6879) Figure; GenBank Accession No. Z50020) ClaI/SwaIYAT1::E389D9eS/EgD8M::Lip1, comprising: (1-3217) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; U.S. patentapplication Pub. No. 2006-0094102-A1); E389D9eS/EgD8M: gene fusioncomprising a codon- optimized Δ9 elongase derived from Eutreptiella sp.CCMP389 (“E389D9eS”), a linker, and the synthetic mutant Δ8 desaturasederived from Euglena gracilis (“EgD8M”) (SEQ ID NO: 49) (labeledindividually as “E389S”, “Linker” and “EgD8M” in Figure; U.S. patentapplication Pub. No. 2008- 0254191-A1); Lip1: Lip1 terminator sequencefrom Yarrowia Lip1 gene (GenBank Accession No. Z50020) SalI/ClaIGPM::EgD5SM::Oct comprising: (11982-1) GPM: Yarrowia lipolytica GPMpromoter (labeled as “GPML” in Figure; U.S. Pat. No. 7,202,356); EgD5SM:Synthetic mutant Δ5 desaturase (SEQ ID NO: 35; U.S. patent Pub. No.2010-0075386-A1), derived from Euglena gracilis (U.S. Pat. No.7,678,560) (labeled as “ED5S” in Figure); OCT: OCT terminator sequenceof Yarrowia OCT gene (GenBank Accession No. X69988) SalI/EcoRI YarrowiaUra3 gene (GenBank Accession No. AJ306421) (11982-10363) EcoRI/PacIEXP1::FmD12S::ACO, comprising: (10363-7666) EXP1: Yarrowia lipolyticaexport protein (EXP1) promoter (labeled as “Exp” in Figure; Intl. App.Pub. No. WO 2006/052870); FmD12S: codon-optimized Δ12 elongase (SEQ IDNO: 51), derived from Fusarium moniliforme (labeled as “FD12S” inFigure; U.S. Pat. No. 7,504,259); Aco: Aco terminator sequence fromYarrowia Aco gene (GenBank Accession No. AJ001300)

The pZKL1-2SR9G85 plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8154U1 according to the General Methods. Thetransformant cells were plated onto MM plates and maintained at 30° C.for 3 to 4 days. Single colonies were re-streaked onto MM plates, andthen inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, resuspended in HGM andthen shaken at 250 rpm/min for 5 days. The cells were subjected to fattyacid analysis, according to the General Methods.

GC analyses showed that most of the selected 96 strains produced40-44.5% EPA of total lipids. Five strains (i.e., #44, #46, #47, #66 and#87) that produced about 44.8%, 45.3%, 47%, 44.6% and 44.7% EPA of TFAswere designated as Y8268, Y8269, Y8270, Y8271 and Y8272, respectively.Knockout of the Lip1 loci (GenBank Accession No. Z50020) was notconfirmed in these EPA strains.

The final genotype of strains Y8268, Y8269, Y8270, Y8271 and Y8272 withrespect to wildtype Yarrowia lipolytica ATCC #20362 was Ura+, Pex3−,unknown 1−, unknown 2−, unknown 3−, unknown 4−, unknown 5−, unknown6−,YAT1::ME3S::Pex16, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,GPAT::EgD9e::Lip2, YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1, YAT1::E389D9eS/EgD8M::Lip1,GPD::FmD12::Pex20, YAT1::FmD12::Oct, EXP1::FmD12S::Aco,EXP1::EgD5M::Pex16, YAT1::EaD5SM::Oct, FBAIN::EgD5SM::Pex20,GPDIN::EgD5SM::Aco, GPM::EgD5SM::Oct, FBAINm::PaD17::Aco,EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1, YAT1::YlCPT::Aco.

Generation of Strain Y8269U (Ura3−)

To disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ ID NO:40;described in Table 15 of U.S. Pat. Appl. Pub. No. 2009-0093543-A1) wasused to integrate a Ura3 mutant gene into the Ura3 gene of strain Y8269,in a manner similar to that described for pZKUM transformation of strainY8006 (supra). A total of 8 transformants were grown and identified topossess a Ura− phenotype.

GC analyses showed that there were 23.0%, 23.1% and 24.2% EPA of TFAs inpZKUM-transformant strains #2, #3 and #5, respectively. These strainswere designated as strains Y8269U1, Y8269U2 and Y8269U3, respectively(collectively, Y8269U).

Generation of Strain Y8406 and Strain Y8412 to Produce about 51.2% EPAand 55.8% EPA of TFAs

Construct pZSCP-Ma83 (FIG. 6B; SEQ ID NO:53) was generated to integrateone Δ8 desaturase gene, one C_(16/18) elongase gene and one malonyl-CoAsynthetase gene into the SCP2 loci (GenBank Accession No. XM_(—)503410)of strain Y8269U1 to thereby enable higher level production of EPA. ThepZSCP-Ma83 plasmid contained the following components:

TABLE 7 Description of Plasmid pZSCP-Ma83 (SEQ ID NO: 53) RE Sites AndNucleotides Within SEQ ID NO: 53 Description Of Fragment And ChimericGene Components BsiWI/AscI 1327 bp 3′ portion of Yarrowia SCP2 gene(labeled as “SCP2-3′” (1-1328) in Figure; GenBank Accession No.XM_503410) SphI/PacI 1780 bp 5′ portion of Yarrowia SCP2 gene (labeledas “SCP2-5′” (4036-5816) in Figure; GenBank Accession No. XM_503410)SwaI/BsiWI GPD::ME3S::Pex20, comprising: (12994-1) GPD: Yarrowialipolytica GPD promoter (U.S. Pat. No. 7,259,255); ME3S: codon-optimizedC_(16/18) elongase gene (SEQ ID NO: 54), derived from M. alpina (U.S.Pat. No. 7,470,532); Pex20: Pex20 terminator sequence from YarrowiaPex20 gene (GenBank Accession No. AF054613) PmeI/SwaI YAT1::MCS::Lip1comprising: (10409-12994) YAT1: Yarrowia lipolytica YAT1 promoter(labeled as “YAT” in Figure; U.S. patent application Pub. No.2006/0094102-A1); MCS: codon-optimized malonyl-CoA synthetase gene (SEQID NO: 56), derived from Rhizobium leguminosarum bv. viciae 3841 (U.S.patent application No. 12/637877); Lip1: Lip1 terminator sequence fromYarrowia Lip1 gene (GenBank Accession No. Z50020) ClaI/PmeIGPD::EaD8S::Pex16 comprising: (7917-10409) GPD: Yarrowia lipolytica GPDpromoter (U.S. Pat. No. 7,259,255); EaD8S: codon-optimized Δ8 desaturasegene (SEQ ID NO: 58), derived from Euglena anabaena (U.S. patentapplication Pub. No. 2008-0254521-A1); Pex16: Pex16 terminator sequencefrom Yarrowia Pex16 gene (GenBank Accession No. U75433) SalI/EcoRIYarrowia Ura3 gene (GenBank Accession No. AJ306421) (7467-5848)

The pZSCP-Ma83 plasmid was digested with AscI/SphI, and then used fortransformation of strains Y8269U1, Y8269U2 and Y8269U3, separately,according to the General Methods. The transformant cells were platedonto MM plates and maintained at 30° C. for 3 to 4 days. Single colonieswere re-streaked onto MM plates, and then inoculated into liquid MM at30° C. and shaken at 250 rpm/min for 2 days. The cells were collected bycentrifugation, resuspended in HGM and then shaken at 250 rpm/min for 5days. The cells were subjected to fatty acid analysis, according to theGeneral Methods.

A total of 96 strains resulting from each pZSCP-Ma83 transformation(i.e., into Y8269U1, Y8269U2 and Y8269U3) were analyzed by GC. Most ofthe selected 288 strains produced 43-47% EPA of TFAs. Seven strains ofY8269U1 transformed with pZSCP-Ma83 (i.e., #59, #61, #65, #67, #70, #81and #94) that produced about 51.3%, 47.9%, 50.8%, 48%, 47.8%, 47.8% and47.8% EPA of TFAs were designated as strains Y8404, Y8405, Y8406, Y8407,Y8408, Y8409 and Y8410, respectively. Three strains of Y8269U2transformed with pZSCP-Ma83 (i.e., #4, #13 and #17) that produced about48.8%, 50.8%, and 49.3% EPA of TFAs were designated as Y8411, Y8412 andY8413, respectively. And, two strains of Y8269U3 transformed withpZSCP-Ma83 (i.e., #2, and #16) that produced about 49.3% and 53.5% EPAof TFAs were designated as Y8414 and Y8415, respectively.

Knockout of the SCP2 loci (GenBank Accession No. XM_(—)503410) was notconfirmed in any of these EPA strains, produced by transformation withpZSCP-Ma83.

The final genotype of strains Y8404, Y8405, Y8406, Y8407, Y8408, Y8409,Y8410, Y8411, Y8412, Y8413, Y8414 and Y8415 with respect to wildtypeYarrowia lipolytica ATCC #20362 was Ura+, Pex3−, unknown 1−, unknown 2−,unknown 3−, unknown 4−, unknown 5−, unknown6−, unknown 7−,YAT1::ME3S::Pex16, GPD::ME3S::Pex20, FBAINm::EgD9eS::Lip2,EXP1::EgD9eS::Lip1, GPAT::EgD9e::Lip2, YAT1::EgD9eS::Lip2,FBAINm::EgD8M::Pex20, EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1,GPD::EaD8S::Pex16, YAT1::E389D9eS/EgD8M::Lip1, GPD::FmD12::Pex20,YAT1::FmD12::Oct, EXP1::FmD12S::Aco, EXP1::EgD5M::Pex16,YAT1::EaD5SM::Oct, FBAIN::EgD5SM::Pex20, GPDIN::EgD5SM::Aco,GPM::EgD5SM::Oct, FBAINm::PaD17::Aco, EXP1::PaD17::Pex16,YAT1::PaD17S::Lip1, YAT1::YlCPT::Aco, YAT1::MCS::Lip1.

Yarrowia lipolytica strain Y8406 was deposited with the American TypeCulture Collection on May 14, 2009 and bears the designation ATCCPTA-10025. Yarrowia lipolytica strain Y8412 was deposited with theAmerican Type Culture Collection on May 14, 2009 and bears thedesignation ATCC PTA-10026.

Analysis of Total Lipid Content and Composition by Flask Assay

Cells from YPD plates of strains Y8404, Y8405, Y8406, Y8407, Y8408,Y8409, Y8410, Y8411, Y8412, Y8413, Y8414 and Y8415 were grown andanalyzed for total lipid content and composition, as follows.

Specifically, one loop of freshly streaked cells was inoculated into 3mL FM medium and grown overnight at 250 rpm and 30° C. The OD_(600nm)was measured and an aliquot of the cells were added to a finalOD_(600nm) of 0.3 in 25 mL FM medium in a 125 mL flask. After 2 days ina shaker incubator at 250 rpm and at 30° C., 6 mL of the culture washarvested by centrifugation and resuspended in 25 mL HGM in a 125 mLflask. After 5 days in a shaker incubator at 250 rpm and at 30° C., a 1mL aliquot was used for fatty acid analysis (supra) and 10 mL dried fordry cell weight [“DCW”] determination.

For DCW determination, 10 mL culture was harvested by centrifugation for5 min at 4000 rpm in a Beckman GH-3.8 rotor in a Beckman GS-6Rcentrifuge. The pellet was resuspended in 25 mL of water andre-harvested as above. The washed pellet was re-suspended in 20 mL ofwater and transferred to a pre-weighed aluminum pan. The cell suspensionwas dried overnight in a vacuum oven at 80° C. The weight of the cellswas determined.

Data from flask assays are presented as Table 8. The Table presents thetotal dry cell weight of the cells [“DCW’], the total lipid content ofcells [“FAME % DCW”], the concentration of each fatty acid as a weightpercent of TFAs [“% TFAs”] and the EPA content as a percent of the drycell weight [“EPA FAME % DCW”]. More specifically, fatty acids will beidentified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0 (stearicacid), 18:1 (oleic acid), 18:2 (LA), ALA, EDA, DGLA, ARA, ETrA, ETA, EPAand other.

TABLE 8 Total Lipid Content And Composition In Yarrowia Strains Y8404,Y8405, Y8406, Y8407, Y8408, Y8409, Y8410, Y8411, Y8412, Y8413, Y8414 AndY8415 By Flask Assay Total EPA DCW FAME % TFAs FAME Strain (g/L) % DCW16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA EtrA ETA EPA other % DCW Y84044.1 27.3 2.8 0.8 1.8 5.1 20.4 2.1 2.9 2.5 0.6 0.8 2.4 51.1 6.3 14.0Y8405 3.9 29.6 2.7 0.5 2.9 5.7 20.5 2.8 2.7 2.1 0.5 0.7 2.0 51.4 5.115.2 Y8406 4.0 30.7 2.6 0.5 2.9 5.7 20.3 2.8 2.8 2.1 0.5 0.8 2.1 51.25.4 15.7 Y8407 4.0 29.4 2.6 0.5 3.0 5.6 20.5 2.8 2.7 2.1 0.4 0.7 2.151.5 5.1 15.2 Y8408 4.1 29.8 2.9 0.6 2.7 5.7 20.2 2.8 2.6 2.1 0.5 0.92.1 51.2 5.5 15.3 Y8409 3.9 30.8 2.8 0.5 2.9 5.7 20.6 2.7 2.7 2.1 0.50.8 2.1 51.0 5.2 15.7 Y8410 4.0 31.8 2.7 0.5 3.0 5.7 20.5 2.9 2.7 2.10.5 0.7 2.1 50.9 5.3 16.2 Y8411 3.6 30.5 2.7 0.3 3.3 5.1 19.9 2.6 2.42.0 0.5 0.6 1.8 52.9 5.7 16.1 Y8412 3.2 27.0 2.5 0.4 2.6 4.3 19.0 2.42.2 2.0 0.5 0.6 1.9 55.8 5.6 15.1 Y8413 2.9 27.2 3.1 0.4 2.6 5.4 19.92.2 2.8 2.0 0.5 0.7 1.8 52.4 5.9 14.2 Y8414 3.7 27.1 2.5 0.7 2.3 6.019.9 1.6 3.4 3.4 0.6 0.6 3.1 49.4 6.1 13.4 Y8415 3.6 25.9 1.4 0.3 1.94.5 16.0 1.3 2.7 2.9 0.5 0.6 2.5 59.0 6.1 15.3Generation of Strain Y8406U (Ura3−)

To disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ ID NO:40;described in Table 15 of U.S. Pat. Appl. Pub. No. 2009-0093543-A1) wasused to integrate a Ura3 mutant gene into the Ura3 gene of strain Y8406in a manner similar to that described for pZKUM transformation of strainY8006 (supra). Several transformants were grown and identified topossess a Ura− phenotype.

GC analyses showed that there were 26.1% EPA of FAMEs inpZKUM-transformant strains #4 and #5. These two strains were designatedas strains Y8406U1 and Y8406U2, respectively (collectively, Y8406U).

Example 2 Generation of Yarrowia lipolytica Strain Y5037 to Produceabout 18.6% EPA, 22.8% DPA and 9.7% DHA of Total Fatty Acids

The present Example describes the construction of strain Y5037, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing about 18.6%EPA, 22.8% DPA and 9.7% DHA relative to the total lipids via expressionof a Δ9 elongase/Δ8 desaturase pathway. This strain was used as theDHA-producing host cell in Example 5.

Briefly, as diagrammed in FIG. 7, strain Y5037 was derived from Yarrowialipolytica ATCC #20362 via construction of strain Y2224 (a FOA resistantmutant from an autonomous mutation of the Ura3 gene of wildtype Yarrowiastrain ATCC #20362), strain Y4001 (producing 17% EDA with a Leu−phenotype), strain Y4001U1 (Leu− and Ura−), strain Y4036 (producing 18%DGLA with a Leu− phenotype), strain Y4036U (Leu− and Ura−), strain Y4070(producing 12% ARA with a Ura− phenotype), strain Y4086 (producing 14%EPA), strain Y4086U1 (Ura3−), strain Y4128 (producing 37% EPA; depositedwith the American Type Culture Collection on Aug. 23, 2007, bearing thedesignation ATCC PTA-8614), strain Y4128U3 (Ura−), strain Y4217(producing 42% EPA), strain Y4217U2 (Ura−), strain Y4259 (producing46.5% EPA), strain Y4259U2 (Ura−), strain Y4305 (producing 53.2% EPA),strain Y4305U3 (Ura−), strain Y5004 (producing 17% EPA, 18.7% DPA and6.4% DHA), strain Y5004U1 (Ura−), strain Y5018 (producing 25.4% EPA,11.4% DPA and 9.4% DHA), strain Y5018U1 (Ura−) and strain Y5037(producing 18.6% EPA, 22.8% DPA and 9.7% DHA relative to the totalTFAs). Further details regarding the construction of strains Y2224,Y4001, Y4001U, Y4036, Y4036U, Y4070, Y4086, Y4086U1, Y4128, Y4128U3,Y4217, Y4217U2, Y4259, Y4259U2, Y4305 and Y4305U3 are described in theGeneral Methods of U.S. Pat. App. Pub. No. 2008-0254191-A1 and inExamples 1-3 of U.S. Pat. App. Pub. No. 2009-0093543-A1, herebyincorporated herein by reference.

The complete lipid profile of strain Y4305 was as follows: 16:0 (2.8%),16:1 (0.7%), 18:0 (1.3%), 18:1 (4.9%), 18:2 (17.6%), ALA (2.3%), EDA(3.4%), DGLA (2.0%), ARA (0.6%), ETA (1.7%), and EPA (53.2%). The totallipid content of cells [“TFAs % DCW”] was 27.5.

The final genotype of strain Y4305 with respect to wild type Yarrowialipolytica ATCC #20362 was SCP2− (YALI0E01298g), YALI0C18711g−, Pex10−,YALI0F24167g−, unknown 1−, unknown 3−, unknown 8−, GPD::FmD12::Pex20,YAT1::FmD12::OCT, GPM/FBAIN::FmD12S::OCT, EXP1::FmD12S::Aco,YAT1::FmD12S::Lip2, YAT1::ME3S::Pex16, EXP1::ME3S::Pex20 (3 copies),GPAT::EgD9e::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2,FBA::EgD9eS::Pex20, GPD::EgD9eS::Lip2, YAT1::EgD9eS::Lip2,YAT1::E389D9eS::OCT, FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1 (2copies), EXP1::EgD8M::Pex16, GPDIN::EgD8M::Lip1, YAT1::EgD8M::Aco,FBAIN::EgD5::Aco, EXP1::EgD5S::Pex20, YAT1::EgD5S::Aco,EXP1::EgD5S::ACO, YAT1::RD5S::OCT, YAT1::PaD17S::Lip1,EXP1::PaD17::Pex16, FBAINm::PaD17::Aco, YAT1::YlCPT1::ACO,GPD::YlCPT1::ACO (wherein FmD12 is a Fusarium moniliforme Δ2 desaturasegene [U.S. Pat. No. 7,504,259]; FmD12S is a codon-optimized Δ2desaturase gene, derived from Fusarium moniliforme [U.S. Pat. No.7,504,259]; MESS is a codon-optimized C_(16/18) elongase gene, derivedfrom Mortierella alpina [U.S. Pat. No. 7,470,532]; EgD9e is a Euglenagracilis Δ9 elongase gene [U.S. Pat. No. 7,645,604]; EgD9eS is acodon-optimized Δ9 elongase gene, derived from Euglena gracilis [U.S.Pat. No. 7,645,604]; E389D9eS is a codon-optimized Δ9 elongase gene,derived from Eutreptiella sp. CCMP389 [U.S. Pat. No. 7,645,604]; EgD8Mis a synthetic mutant Δ8 desaturase [U.S. Pat. No. 7,709,239], derivedfrom Euglena gracilis [U.S. Pat. No. 7,256,033]; EgD5 is a Euglenagracilis Δ5 desaturase [U.S. Pat. No. 7,678,560]; EgD5S is acodon-optimized Δ5 desaturase gene, derived from Euglena gracilis [U.S.Pat. No. 7,678,560]; RD5S is a codon-optimized Δ5 desaturase, derivedfrom Peridinium sp. CCMP626 [U.S. Pat. No. 7,695,950]; PaD17 is aPythium aphanidermatum Δ17 desaturase [U.S. Pat. No. 7,556,949]; PaD17Sis a codon-optimized Δ17 desaturase, derived from Pythium aphanidermatum[U.S. Pat. No. 7,556,949]; and, YlCPT1 is a Yarrowia lipolyticadiacylglycerol cholinephosphotransferase gene [Intl. App. Pub. No. WO2006/052870]).

Strain Y4305U (Ura3−) was generated via integrating a Ura3 mutant geneinto the Ura3 gene of strain Y4305.

Generation of Y5004 Strain to Produce about 17.0% EPA, 18.7% DPA and6.4% DHA of TFAs

Construct pZKL4-220EA41B (FIG. 8A; SEQ ID NO:60) was constructed tointegrate two C₂₀₋₂₂ elongase genes and two Δ4 desaturase genes into thelipase 4-like locus (GenBank Accession No. XM_(—)503825) of strainY4305U3. The pZKL4-220EA41B plasmid contained the following components:

TABLE 9 Components Of Plasmid pZKL4-220EA41B (SEQ ID NO: 60) RE SitesAnd Nucleotides Within SEQ ID NO: 60 Description Of Fragment AndChimeric Gene Components AscI/BsiWI 745 bp 5′ portion of the YarrowiaLipase 4-like gene (GenBank (9777-9025) Accession No. XM_503825; labeledas “Lip4” in Figure) PacI/SphI 782 bp 3′ portion of Yarrowia Lipase 4like gene (GenBank (13273-12485) Accession No. XM_503825; labeled as“Lip4-3′” in Figure) SwaI/BsiWI FBAINm::EaC20ES::Pex20, comprising:(6882-9025) FBAINm: Yarrowia lipolytica FBAINm promoter (U.S. Pat. No.7,202,356) EaC20ES: codon-optimized C20 elongase gene (SEQ ID NO: 61),derived from Euglena anabaena (U.S. patent application Pub. No.2008/0254191-A1); Pex20: Pex20 terminator sequence from Yarrowia Pex20gene (GenBank Accession No. AF054613) PmeI/SwaI YAT1::EgC20ES::Lip1,comprising: (4903-6882) YAT1: Yarrowia lipolytica YAT1 promoter (U.S.patent application Pub. No. 2006/0094102-A1); EgC20ES: codon-optimizedC20 elongase gene (SEQ ID NO: 63), derived from Euglena gracilis (U.S.patent application Pub. No. 2008/0254191-A1); Lip1: Lip1 terminatorsequence from Yarrowia Lip1 gene (GenBank Accession No. Z50020)PmeI/ClaI EXP1::EaD4S-1::Lip2, comprising: (4903-2070) EXP1: Yarrowialipolytica export protein (EXP1) promoter (Intl. App. Pub. No. WO2006/052870); EaD4S-1: codon-optimized truncated Δ4 desaturase (SEQ IDNO: 65), derived from Euglena anabaena (U.S. patent application Pub. No.2008/0254191-A1); Lip2: Lip2 terminator sequence from Yarrowia Lip2 gene(GenBank Accession No. AJ012632) SalI/EcoRI Yarrowia Ura3 gene (GenBankAccession No. AJ306421) (1620-1) EcoRI/PacI GPDIN::EaD4SB::Aco,comprising: (1-14039) GPDIN: Yarrowia lipolytica GPDIN promoter (U.S.Pat. No. 7,459,546); EaD4SB: codon-optimized truncated Δ4 desaturaseversion B (SEQ ID NO: 67), derived from Euglena anabaena (U.S. patentapplication Pub. No. 2008/0254191-A1); Aco: Aco terminator sequence fromYarrowia Aco gene (GenBank Accession No. AJ001300)

The pZKL4-220EA41B plasmid was digested with AscI/SphI, and then usedfor transformation of strain Y4305U3 (supra), according to the GeneralMethods. The transformants were selected on MM plates. After days growthat 30° C., 72 transformants grown on the MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into 3 mL liquid MM at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation,resuspended in HGM and then shaken at 250 rpm/min for 5 days. The cellswere subjected to fatty acid analysis, according to the General Methods.

GC analyses showed the presence of DHA in the transformants withpZKL4-220EA41B, but not in the parent Y4305U strain. Most of theselected 72 strains produced about 22% EPA, 18% DPA and 5% DHA of TFAs.Strain #2 produced 17% EPA, 18.7% DPA and 6.4% DHA, while strain #33produced 21.5% EPA, 21% DPA and 5.5% DHA. These two strains weredesignated as Y5004 and Y5005, respectively.

Knockout of the lipase 4-like locus (GenBank Accession No. XM_(—)503825)was not confirmed in either strain Y5004 or Y5005.

Generation of Strain Y5004U (Ura3−)

To disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ ID NO:40;described in Table 15 of U.S. Pat. App. Pub. No. 2009-0093543-A1) wasused to integrate a Ura3 mutant gene into the Ura3 gene of strain Y5004,in a manner similar to that described for pZKUM transformation of strainY8006 (Example 1).

A total of 8 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. All 8strains had a Ura− phenotype (i.e., cells could grow on MM+5-FOA plates,but not on MM plates). The cells were scraped from the MM+5-FOA platesand subjected to fatty acid analysis, according to the General Methods.

GC analyses showed the presence of 14.8% EPA, 17.4% DPA and 0.4% DHA ofTFAs in transformant #5 and 15.3% EPA, 17.2% DPA and 0.4% DHA of TFAs intransformant #8. These two strains were designated as strains Y5004U1and Y5004U2, respectively (collectively, Y5004U).

Generation of Strain Y5018 to Produce about 25.4% EPA, 11.4% DPA and9.4% DHA of TFAs

Construct pZKL3-4GER44 (FIG. 8B; SEQ ID NO:69) was constructed tointegrate one C_(20/22) elongase gene and three Δ4 desaturase genes intothe lipase 3-like locus (GenBank Accession No. XP_(—)506121) of strainY5004U1. The pZKL3-4GER44 plasmid contained the following components:

TABLE 10 Components Of Plasmid pZKL3-4GER44 (SEQ ID NO: 69) RE Sites AndNucleotides Within SEQ ID NO: 69 Description Of Fragment And ChimericGene Components AscI/BsiWI 887 bp 5′ portion of the Yarrowia Lipase3-like gene (GenBank (10527-9640) Accession No. XP_506121) PacI/SphI 804bp 3′ portion of Yarrowia Lipase 3-like gene (GenBank (14039-13235)Accession No. XP_506121) SwaI/BsiWI FBAINm::EgC20ES::Pex20, comprising:(7485-9640) FBAINm: Yarrowia lipolytica FBAINm promoter (U.S. Pat. No.7,202,356); EgC20ES: codon-optimized C20 elongase gene (SEQ ID NO: 63),derived from Euglena gracilis (U.S. patent application Pub. No.2008/0254191-A1); Pex20: Pex20 terminator sequence from Yarrowia Pex20gene (GenBank Accession No. AF054613) PmeI/SwaI YAT1::EaD4S-1::Lip1,comprising: (4774-7485) YAT1: Yarrowia lipolytica YAT1 promoter (U.S.patent application Pub. No. 2006/0094102-A1); EaD4S-1: codon-optimizedtruncated Δ4 desaturase (SEQ ID NO: 65), derived from Euglena anabaena(U.S. patent application Pub. No. 2008/0254191-A1); Lip1: Lip1terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.Z50020) ClaI/PmeI EXP1::E1594D4S::Oct, comprising: (2070-4774) EXP1:Yarrowia lipolytica export protein promoter (Intl. App. Pub. No. WO2006/052870); E1594D4S: codon-optimized Δ4 desaturase (SEQ ID NO: 70),derived from Eutreptiella cf_gymnastica CCMP1594 (U.S. patentapplication Pub. No. 2009/0253188-A1) (labeled as “D4S-1594” in Figure);OCT: OCT terminator sequence of Yarrowia OCT gene (GenBank Accession No.X69988) SalI/EcoRI Yarrowia Ura3 gene (GenBank Accession No. AJ306421)(1620-1) EcoRI/PacI GPDIN::EgD4S-1::Aco, comprising: (1-14039) GPDIN:Yarrowia lipolytica GPDIN promoter (U.S. Pat. No. 7,459,546); EgD4S-1:codon-optimized truncated Δ4 desaturase (SEQ ID NO: 72), derived fromEuglena gracilis (U.S. patent application Pub. No. 2008/0254191-A1);Aco: Aco terminator sequence from Yarrowia Aco gene (GenBank AccessionNo. AJ001300)

The pZKL3-4GER44 plasmid was digested with AscI/SphI, and then used fortransformation of strain Y5004U1, according to the General Methods. Thetransformants were selected on MM plates. After 5 days growth at 30° C.,96 transformants grown on the MM plates were picked and re-streaked ontofresh MM plates. Once grown, these strains were individually inoculatedinto 3 mL liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. Thecells were collected by centrifugation, resuspended in HGM and thenshaken at 250 rpm/min for 5 days. The cells were subjected to fatty acidanalysis, according to the General Methods.

GC analyses showed that most of the selected 96 strains produced about19% EPA, 22% DPA and 7% DHA of TFAs. Strain #1 produced 23.3% EPA, 13.7%DPA and 8.9% DHA, while strain #49 produced 25.2% EPA, 11.4% DPA and9.4% DHA. These two strains were designated as Y5011 and Y5018,respectively.

Knockout of the lipase 3-like locus (GenBank Accession No. XP_(—)506121)was not confirmed in strains Y5011 and Y5018.

Generation of Strain Y5018U (Ura3−)

To disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ ID NO:40;described in Table 15 of U.S. Pat. App. Pub. No. 2009-0093543-A1) wasused to integrate a Ura3 mutant gene into the Ura3 gene of strain Y5018,in a manner similar to that described for pZKUM transformation of strainY8006 (Example 1). A total of 18 transformants were grown and identifiedto possess a Ura− phenotype.

GC analyses showed the presence of 16.6% EPA, 10.4% DPA and 0.0% DHA ofFAMEs in pZKUM-transformant strain #2 and 17.0% EPA, 10.8% DPA and 0.0%DHA of FAMEs in pZKUM-transformant strain #4. These two strains weredesignated as strains Y5018U1 and Y5018U2, respectively (collectively,Y5018U).

Generation of Strain Y5037 to Produce about 18.6% EPA, 22.8% DPA and9.7% DHA of TFAs

Construct pZKLY-G20444 (FIG. 9; SEQ ID NO:74) was constructed tointegrate one DHA synthase and two Δ4 desaturase genes into the lipase7-like locus (GenBank Accession No. AJ549519) of strain Y5018U1. A DHAsynthase is a multizyme comprising a C20 elongase linked to a Δ4desaturase. The pZKLY-G20444 plasmid contained the following components:

TABLE 11 Components Of Plasmid pZKLY-G20444 (SEQ ID NO: 74) RE Sites AndNucleotides Within SEQ ID NO: 74 Description Of Fragment And ChimericGene Components AscI/BsiWI 887 bp 5′ portion of the Yarrowia Lipase7-like gene (labeled as (9370-8476) “LipY-5′” in Figure; GenBankAccession No. AJ549519) PacI/SphI 756 bp 3′ portion of Yarrowia Lipase7-like gene (labeled as (12840-12078) “LipY-3′” in Figure; GenBankAccession No. AJ549519) PmeI/SwaI YAT1::EgDHAsyn1S::Lip1, comprising:(4871-8320) YAT1: Yarrowia lipolytica YAT1 promoter (U.S. patentapplication Pub. No. 2006/0094102-A1); EgDHAsyn1S: codon-optimized DHAsynthase (SEQ ID NO: 75), derived from Euglena gracilis (labeled as“EgDHAase” in Figure; U.S. patent application Pub. No. 2008/0254191-A1);Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene (GenBankAccession No. Z50020) ClaI/PmeI EXP1::EaD4S-1::Pex16, comprising:(2070-4871) EXP1: Yarrowia lipolytica export protein (EXP1) promoter(Intl. App. Pub. No. WO 2006/052870); EaD4S-1: codon-optimized truncatedΔ4 desaturase (SEQ ID NO: 65), derived from Euglena anabaena (U.S.patent application Pub. No. 2008/0254191-A1); Pex16: Pex16 terminatorsequence from Yarrowia Pex16 gene (GenBank Accession No. U75433)SalI/EcoRI Yarrowia Ura3 gene (GenBank Accession No. AJ306421) (1620-1)EcoRI/PmeI FBAINm::E1594D4S::Pex16, comprising: (1-12871) FBAINm:Yarrowia lipolytica FBAINm promoter (U.S. Pat. No. 7,202,356); E1594D4S:codon-optimized Δ4 desaturase (SEQ ID NO: 70), derived from Eutreptiellacf_gymnastica CCMP1594 (U.S. patent application Pub. No.2009/0253188-A1) (labeled as “D4S-1594” in Figure); Pex16: Pex16terminator sequence from Yarrowia Pex16 gene (GenBank Accession No.U75433)

The pZKLY-G20444 plasmid was digested with AscI/SphI, and then used fortransformation of strain Y5018U1, according to the General Methods. Thetransformants were selected on MM plates. After 5 days growth at 30° C.,96 transformants grown on the MM plates were picked and re-streaked ontofresh MM plates. Once grown, these strains were individually inoculatedinto 3 mL liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. Thecells were collected by centrifugation, resuspended in HGM and thenshaken at 250 rpm/min for 5 days. The cells were subjected to fatty acidanalysis, according to the General Methods.

GC analyses showed that most of the selected 96 strains produced about19% EPA, 22% DPA and 9% DHA of TFAs. Strain #3 produced 18.6% EPA, 22.8%DPA and 9.7% DHA; strain #9 produced 18.4% EPA, 21% DPA and 9.6% DHA;strain #27 produced 17.8% EPA, 20.6% DPA and 10% DHA; and strain #40produced 18.8% EPA, 21.2% DPA and 9.6% DHA. These four strains weredesignated as Y5037, Y5038, Y5039 and Y5040, respectively.

Knockout of the lipase 7-like locus (GenBank Accession No, AJ549519) wasnot confirmed in these knocked out strains.

The final genotype of strains Y5037, Y5038, Y5039 and Y5040 with respectto wild type Yarrowia lipolytica ATCC #20362 was SCP2−(YALI0E01298g),YALI0C18711g−, Pex10−, YALI0F24167g−, unknown 1−, unknown 3−, unknown8−, unknown 9−, unknown10−, unknown 11−, GPD::FmD12::Pex20,YAT1::FmD12::OCT, GPM/FBAIN::FmD12S::OCT, EXP1::FmD12S::Aco,YAT1::FmD12S::Lip2, YAT1::ME3S::Pex16, EXP1::ME3S::Pex20 (3 copies),GPAT::EgD9e::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2,FBA::EgD9eS::Pex20, GPD::EgD9eS::Lip2, YAT1::EgD9eS::Lip2,YAT1::E389D9eS::OCT, FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1 (2copies), EXP1::EgD8M::Pex16, GPDIN::EgD8M::Lip1, YAT1::EgD8M::Aco,FBAIN::EgD5::Aco, EXP1::EgD5S::Pex20, YAT1::EgD5S::Aco,EXP1::EgD5S::ACO, YAT1::RD5S::OCT, YAT1::PaD17S::Lip1,EXP1::PaD17::Pex16, FBAINm::PaD17::Aco, YAT1::YlCPT1::ACO,GPD::YlCPT1::ACO, FBAINm::EaC20ES::Pex20, YAT1::EgC20ES::Lip1,FBAINm::EgC20ES::Pex20, EXP1::EaD4S-1::Lip2, EXP1::EaD4S-1::Pex16,YAT1::EaD4S-1::Lip1, GPDIN::EaD4SB::Aco, EXP1::E1594D4S::Oct,FBAINm::E1594D4S::Pex16, GPDIN::EgD4S-1::Aco, YAT1::EgDHAsyn1S::Lip1.

Generation of Strain Y5037U (Ura3−)

To disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ ID NO:40;described in Table 15 of U.S. Pat. App. Pub. No. 2009-0093543-A1) wasused to integrate a Ura3 mutant gene into the Ura3 gene of strain Y5037,in a manner similar to that described for pZKUM transformation of strainY5004 (supra). A total of 12 transformants were grown and identified topossess a Ura− phenotype.

GC analyses showed the presence of 12.1% EPA, 10.2% DPA and 3.3% DHA inpZKUM-transformant strain #4 and 12.4% EPA, 10.3% DPA and 3.5% DHA inpZKUM-transformant strain #11. These two strains were designated asstrains Y5037U1 and Y5037U2, respectively (collectively, Y5037U).

Example 3 Construction of Various Expression Vectors ComprisingDifferent LPLAT ORFs

The present example describes the construction of a series of vectors,each comprising a LPLAT ORF, suitable for expression in Yarrowialipolytica. LPLAT ORFs included the Saccharomyces cerevisiae Ale1,Yarrowia lipolytica Ale1, Mortierella alpina LPAAT1, Yarrowia lipolyticaLPAAT1 and Caenorhabditis elegans LPCAT. Examples 4, 5 and 6 describethe results obtained following transformation of these vectors intoYarrowia lipolytica.

Origin of LPLATs

A variety of LPLATs have been identified in the patent and openliterature, but the functionality of these genes has not been previouslydirectly compared. Table 12 summarizes publicly available LPLATs (i.e.,ScAle1, ScLPAAT, MaLPAAT1 and CeLPCAT) and LPLAT orthologs identifiedherein (i.e., YlAle1 and YlLPAAT1) that are utilized in the Examples,following codon-optimization of heterologous genes for expression inYarrowia lipolytica (infra).

TABLE 12 LPLATs Functionally Characterized ORF SEQ ID LPLAT OrganismDesignation References NO Ale1 Saccharomyces ORF GenBank Accession No.8, 9 cerevisiae* “YOR175C” or NP_014818; U.S. patent “ScAle1”application Pub. No. 20080145867 (and corresponding to Intl. App. Pub.No. WO 2008/076377); Intl. App. Pub. No. WO 2009/001315 Yarrowia“YALI0F19514p” GenBank Accession No. 10, 11 lipolytica or “YlAle1”XP_505624; Intl. App. Pub. No. WO 2009/001315 LPAAT Saccharomyces ORF“YDL052C” GenBank Accession No. 18 cerevisiae or “ScLPAAT” NP_010231Mortierella “MaLPAAT1” U.S. patent application Pub. 14, 15 alpina No.2006-0115881-A1; U.S. patent application Pub. No. 2009-0325265-A1Yarrowia “YALI0E18964g” GenBank Accession No. 16, 17 lipolytica or“YlLPAAT1” XP_504127; U.S. Pat. No. 7,189,559 LPCAT Caenorhabditis“clone T06E8.1” GenBank Accession No. 1, 2 elegans* or “CeLPCAT”CAA98276; Intl. App. Pub. No. WO 2004/076617 (corresponding to U.S.patent application Pub. No. 2006- 0168687-A1) *The Saccharomycescerevisiae Ale1 and Caenorhabditis elegans LPCAT were used ascomparative Examples.

More specifically, the ScLPAAT (SEQ ID NO:18) and ScAle1 (SEQ ID NO:9)protein sequences were used as queries to identify orthologs from thepublic Y. lipolytica protein database of the “Yeast project Genolevures”(Center for Bioinformatics, LaBR1, Talence Cedex, France) (see alsoDujon, B. et al., Nature, 430(6995):35-44 (2004)) using the WashingtonUniversity in St. Louis School of Medicine BLAST 2.0 (WU-BLAST;http://blast.wustl.edu). Based on analysis of the best hits, the Ale1and LPAAT orthologs from Yarrowia lipolytica are identified herein asYlAle1 (SEQ ID NO:11) and YlLPAAT (SEQ ID NO:17), respectively. Theidentity of YlAle1 and YlLPAAT1 as orthologs of ScAle1 and ScLPAAT,respectively, was further confirmed by doing a reciprocal BLAST, i.e.,using SEQ ID NOs:11 and 17 as a query against the Saccharomycescerevisiae public protein database to find ScAle1 and ScLPAAT,respectively, as the best hits.

The LPLAT proteins identified above as ScAle1 (SEQ ID NO:9), YlAle1 (SEQID NO:11), ScLPAAT (SEQ ID NO:18), MaLPAAT1 (SEQ ID NO:15), YlLPAAT1(SEQ ID NO:17) and CeLPCAT (SEQ ID NO:2) were aligned using the methodof Clustal W (slow, accurate, Gonnet option; Thompson et al., NucleicAcids Res., 22:4673-4680 (1994)) of the MegAlign™ program (version8.0.2) of the LASERGENE bioinformatics computing suite (DNASTAR, Inc.,Madison, Wis.). This resulted in creation of Table 13, where percentsimilarity is shown in the upper triangle of the Table while percentdivergence is shown in the lower triangle.

TABLE 13 Percent Identity And Divergence Among Various LPLATs YlLPAAT1CeLPCAT MaLPAAT1 ScAle1 ScLPAAT YlAle1 —  26.6  34.0  9.6 43.9 11.7YlLPAAT1 184.3 —  36.4 11.3 32.4 14.5 CeLPCAT 137.5 126.4 — 11.1 34.615.0 MaLPAAT1 545.0 442.0 456.0 — 13.5 45.0 ScAle1  97.9 145.7 134.5365.0  — 15.6 ScLPAAT 426.0 339.0 330.0 94.3 317.0  — YlAle1

The percent identities revealed by this method allowed determination ofthe minimum percent identity between each of the LPAAT polypeptides andthe minimum percent identity between each of the Ale1 polypeptides. Therange of identity between LPAAT polypeptides was 34.0% identity(MaLPAAT1 and YlLPAAT1) to 43.9% identity (ScLPAAT and YlLPAAT1), whileidentity between the ScAle1 and YlAle1 polypeptides was 45%.

Membrane Bound O-Acyltransferase [“MBOAT”] Family Motifs:

Orthologs of the ScAle1 protein sequence (SEQ ID NO:9) were identifiedby conducting a National Center for Biotechnology Information [“NCBI”]BLASTP 2.2.20 (protein-protein Basic Local Alignment Search Tool;Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997); and Altschulet al., FEBS J., 272:5101-5109 (2005)) search using ScAle1 (SEQ ID NO:9)as the query sequence against fungal proteins in the “nr” proteindatabase (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure from BrookhavenProtein Data Bank [“PDB”], sequences included in the last major releaseof the SWISS-PROT protein sequence database, PIR and PRF excluding thoseenvironmental samples from WGS projects) using default parameters(expect threshold=10; word size=3; scoring parameters matrix=BLOSUM62;gap costs: existence=11, extension=1). The following hits were obtained:

TABLE 14 Fungal Orthologs Of ScAle1 (SEQ ID NO: 9) Based On BLASTAnalysis Gen Bank Acession No. Species NP_014818.1 Saccharomycescerevisiae XP_001643411.1 Vanderwaltozyma polyspora DSM 70294XP_448977.1 Candida glabrata XP_455985.1 Kluyveromyces lactisNP_986937.1 Ashbya gossypii ATCC 10895 XP_001385654.2 Pichia stipitisCBS 6054 XP_001487052.1 Pichia guilliermondii ATCC 6260 EDK36331.2Pichia guilliermondii ATCC 6260 XP_001525914.1 Lodderomyces elongisporusNRRL YB-4239 XP_461358.1 Debaryomyces hansenii CBS767 XP_713184.1Candida albicans SC5314 XP_001645053.1 Vanderwaltozyma polyspora DSM70294 XP_505624.1 Yarrowia lipolytica XP_001805526.1 Phaeosphaerianodorum SN15 XP_001598340.1 Sclerotinia sclerotiorum 1980 XP_001907785.1Podospora anserine XP_001931658.1 Pyrenophora tritici-repentis Pt-1C-BFPXP_001560657.1 Botryotinia fuckeliana B05.10 XP_963006.1 Neurosporacrassa OR74A XP_364011.2 Magnaporthe grisea 70-15 XP_001209647.1Aspergillus terreus NIH2624 XP_001822945.1 Aspergillus oryzae RIB40XP_001257694.1 Neosartorya fischeri NRRL 181 XP_747591.2 Aspergillusfumigatus Af293 XP_001270060.1 Aspergillus clavatus NRRL 1 NP_596779.1Schizosaccharomyces pombe XP_001396584.1 Aspergillus nigerXP_001229385.1 Chaetomium globosum CBS 148.51 XP_001248887.1Coccidioides immitis RS XP_664134.1 Aspergillus nidulans FGSC A4XP_566668.1 Cryptococcus neoformans var. neoformans JEC21 XP_001839338.1Coprinopsis cinerea okayama 7#130 XP_757554.1 Ustilago maydis 521The yeast and fungal protein sequences of Table 14 were aligned usingDNASTAR. Multiple sequence alignments and percent identity calculationswere performed using the Clustal W method of alignment (supra).

More specifically, default parameters for multiple protein alignmentusing the Clustal W method of alignment correspond to: GAP PENALTY=10,GAP LENGTH PENALTY=0.2, Delay Divergent Seqs(%)=30, DNA TransitionWeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUBwith the ‘slow-accurate’ option. The resulting alignment was analyzed todetermine the presence or absence of the non-plant motifs for Ale1homologs, as identified in U.S. Pat. Pub. No. 2008-0145867-A1.Specifically, these include: M-[V/I]-[L/I]-xxK-[L/V/I]-xxxxxxDG (SEQ IDNO:26), RxKYYxxWxxx-[E/D]-[A/G]xxxxGxG-[F/Y]-xG (SEQ ID NO:27),EX₁₁WNX₂-[T/V]-X₂W (SEQ ID NO:28) and SAxWHGxxPGYxx-[T/F]-F (SEQ IDNO:29), wherein X encodes any amino acid residue. The H is residue inSEQ ID NO:29 has been reported to be a likely active site residue withinthe protein.

Only one motif, i.e., EX₁₁WNX₂-[T/V]-X₂W (SEQ ID NO:28), was completelyconserved in all 33 of the organisms aligned. The remainingM-[V/I]-[L/I]-xxK-[L/V/I]-xxxxxxDG (SEQ ID NO:26),RxKYYxxWxxx-[E/D]-[A/G]xxxxGxG-[F/Y]-xG (SEQ ID NO:27) andSAxWHGxxPGYxx-[T/F]-F (SEQ ID NO:29) motifs were only partiallyconserved. Thus, these motifs were appropriately truncated to fit with 0mismatch (i.e., SAxWHG [SEQ ID NO:5]), 1 mismatch (i.e., RxKYYxxW [SEQID NO:4]), or 2 mismatches (i.e., M(V/I)(L/I)xxK(L/I) [SEQ ID NO:3]) forthe purposes of the present methodologies.

1-Acyl-Sn-Glycerol-3-Phosphate Acyltransferase [“LPAAT”] Family Motifs:

Analysis of the protein alignment comprising ScLPAAT (SEQ ID NO:18),MaLPAAT1 (SEQ ID NO:15) and YlLPAAT1 (SEQ ID NO:17) revealed that the1-acyl-sn-glycerol-3-phosphate acyltransferase family motif EGTR (SEQ IDNO:20) was present in each of the LPAAT orthologs. On this basis,MaLPAAT1 was identified as a likely LPAAT, that was clearlydistinguishable from the Ma LPAAT-like proteins disclosed in Intl. App.Pub. No. WO 2004/087902 (i.e., SEQ ID NOs:93 and 95).

It is noteworthy that the EGTR (SEQ ID NO:20) motif, while lacking inthe LPCAT sequences in Intl. App. Pub. No. WO 2004/087902, is present inCeLPCAT (SEQ ID NO:2). It appears that other residues distinguish LPAATand LPCAT sequences in LPAAT-like proteins. One such residue could bethe extension of the EGTR (SEQ ID NO:20) motif. Specifically, whereasthe EGTR motif in ScLPAAT (SEQ ID NO:18), MaLPAAT1 (SEQ ID NO:15) andYlLPAAT1 (SEQ ID NO:17) is immediately followed by a serine residue, theEGTR motif in CeLPCAT is immediately followed by an asparagine residue.In contrast, the two LPCATs in Intl. App. Pub. No. WO 2004/087902 have avaline substituted for the arginine residue in the EGTR motif and themotif is immediately followed by a valine residue.

Construction of pY201, Comprising a Codon-Optimized Saccharomycescerevisiae Ale1 Gene

The Saccharomyces cerevisiae ORF designated as “ScAle1” (SEQ ID NO:8)was optimized for expression in Yarrowia lipolytica, by DNA 2.0 (MenloPark, Calif.). In addition to codon optimization, 5′ Pci1 and 3′ Not1cloning sites were introduced within the synthetic gene (i.e., ScAle1S;SEQ ID NO:12). None of the modifications in the ScAle1S gene changed theamino acid sequence of the encoded protein (i.e., the protein sequenceencoded by the codon-optimized gene [i.e., SEQ ID NO:13] is identical tothat of the wildtype protein sequence [i.e., SEQ ID NO:9]). ScAle1S wascloned into pJ201 (DNA 2.0) to result in pJ201:ScAle1S.

A 1863 bp Pci1/Not1 fragment comprising ScAle1S was excised frompJ201:ScAle1S and used to create pY201 (SEQ ID NO:77; Table 15; FIG.10A). In addition to comprising a chimeric YAT1::ScAle1S::Lip1 gene,pY201 also contains a Yarrowia lipolytica URA3 selection marker flankedby LoxP sites for subsequent removal, if needed, by Crerecombinase-mediated recombination. Both the YAT1::ScAle1S::Lip1chimeric gene and the URA3 gene were flanked by fragments havinghomology to 5′ and 3′ regions of the Yarrowia lipolytica Pox3 gene tofacilitate integration by double homologous recombination, althoughintegration into Yarrowia lipolytica is known to usually occur withouthomologous recombination. Thus, construct pY201 thereby contained thefollowing components:

TABLE 15 Description of Plasmid pY201 (SEQ ID NO: 77) RE Sites AndNucleotides Within Description Of Fragment And Chimeric Gene SEQ ID NO:77 Components BsiW1/Sbf1 LoxP::Ura3::LoxP, comprising: (1-1706 bp) LoxPsequence (SEQ ID NO: 78) Yarrowia lipolytica Ura3 gene (GenBankAccession No. AJ306421); LoxP sequence (SEQ ID NO: 78) Sbf1/Sph1 3′portion of Yarrowia lipolytica POX3 Acyl-CoA (1706-3043 bp) oxidase 3(GenBank Accession No. YALI0D24750g) (i.e., bp 2215-3038 in pY201)Sph1/Asc1 ColE1 plasmid origin of replication; (3043-5743 bp)Ampicillin-resistance gene (Amp^(R)) for selection in E. coli (i.e., bp3598-4758 [complementary] in pY201); E. coli f1 origin of replicationAscI/BsiWI 5′ portion of Yarrowia lipolytica POX3 Acyl-CoA (5743-6513bp) oxidase 3 (GenBank Accession No. YALI0D24750g) (i.e., bp 5743-6512in pY201) BsiWI/BsiWI YAT1::ScAle1S::Lip1, comprising: (6514-1 bp) YAT1:Yarrowia lipolytica YAT1 promoter (U.S. [a Not1 site, patent applicationPub. No. 2006/0094102-A1) located between (i.e., bp 6514-7291 in pY201)ScAle1S and Lip1 ScAle1S: codon-optimized Ale1 (SEQ ID NO: 12) ispresent at bp derived from Saccharomyces cerevisiae YOR175C 9154 bp](i.e., bp 7292-9151 in pY201; labeled as “Sc LPCATs ORF” in Figure);Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene (GenBankAccession No. Z50020) (i.e., bp 9160-9481 pY201; labeled as “Lip1-3′” inFigure)Construction of pY168, Comprising a Yarrowia lipolytica Ale1 Gene

The Yarrowia lipolytica ORF designated as “YlAle1” (GenBank AccessionNo. XP_(—)505624; SEQ ID NO:10) was amplified by PCR from Yarrowialipolytica ATCC #20362 cDNA library using PCR primers 798 and 799 (SEQID NOs:79 and 80, respectively). Additionally, the YAT promoter wasamplified by PCR primers 800 and 801 (SEQ ID NOs:81 and 82,respectively) from pY201 (SEQ ID NO:77). Since the primer pairs weredesigned to create two PCR products having some overlap with oneanother, a YAT1::YlAle1 fusion fragment was then amplified byoverlapping PCR using primers 798 and 801 (SEQ ID NOs:79 and 82,respectively) and the two PCR fragments as template. The PCR was carriedout in a RoboCycler Gradient 40 PCR machine (Stratagene) using themanufacturer's recommendations and Pfu Ultra™ High-Fidelity DNAPolymerase (Stratagene, Cat. No. 600380). Amplification was carried outas follows: initial denaturation at 95° C. for 4 min, followed by 30cycles of denaturation at 95° C. for 30 sec, annealing at 55° C. for 1min, and elongation at 72° C. for 1 min. A final elongation cycle at 72°C. for 10 min was carried out, followed by reaction termination at 4° C.

The PCR product comprising the YAT1::Yl Ale1 fusion fragment was gelpurified and digested with ClaI/NotI. This Cla1-Not1 fragment wasligated into pY201 that had been similarly digested (thereby removingthe YAT1::ScAle1S fragment) to create pY168 (SEQ ID NO:83), comprising achimeric YAT1::YlAle1::Lip1 gene. The DNA sequence of the Yarrowia Ale1ORF was confirmed by DNA sequencing. The components present in pY168(FIG. 10B; SEQ ID NO:83) are identical to those present in pY201, withthe exception of the YAT1::YlAle1::Lip1 gene in pY168, instead of theYAT1::ScAle1S::Lip1 gene in pY201 (FIG. 10A). Note that YlAle1 islabeled as “Yl LPCAT” in FIG. 10B.

Construction of pY208, Comprising a Mortierella alpina LPAAT1 Gene

The Mortierella alpina ORF designated as “MaLPAAT1” (SEQ ID NO:14) wasoptimized for expression in Yarrowia lipolytica, by DNA 2.0 (Menlo Park,Calif.). In addition to codon optimization, 5′ Pci1 and 3′ Not1 cloningsites were introduced within the synthetic gene (i.e., MaLPAAT1S; SEQ IDNO:21). None of the modifications in the MaLPAAT1S gene changed theamino acid sequence of the encoded protein (i.e., the protein sequenceencoded by the codon-optimized gene [i.e., SEQ ID NO:22] is identical tothat of the wildtype protein sequence [i.e., SEQ ID NO:15]). MaLPAAT1Swas cloned into pJ201 (DNA 2.0) to result in pJ201:MaLPAAT1S.

A 945 bp Pci1/Not1 fragment comprising MaLPAAT1S was excised frompJ201:MaLPAAT1S and used to create pY208 (SEQ ID NO:84), in a 3-wayligation with two fragments of pY201 (SEQ ID NO:77). Specifically, theMaLPAAT1 fragment was ligated with a 3530 bp Sph-Not1 pY201 fragment anda 4248 bp NcoI-SphI pY201 fragment to result in pY208. The componentspresent in pY208 (FIG. 11A; SEQ ID NO:84) are identical to those presentin pY201, with the exception of the YAT1::MaLPAAT1S::Lip1 gene in pY208,instead of the YAT1::Sc Ale1S::Lip1 gene in pY201 (FIG. 10A).

Construction of pY207, Comprising a Yarrowia lipolytica LPAAT1 Gene

A putative LPAAT1 from Yarrowia lipolytica (designated herein as“YlLPAAT1”; SEQ ID NO:17) was described in U.S. Pat. No. 7,189,559 andGenBank Accession No. XP_(—)504127. The protein is annotated as “similarto uniprot|P33333 Saccharomyces cerevisiae YDL052c SLC1 fattyacyltransferase”.

The YlLPAAT1 ORF (SEQ ID NO:16) was amplified by PCR using Yarrowialipolytica ATCC #20362 cDNA library as a template and PCR primers 856and 857 (SEQ ID NOs:85 and 86, respectively). The PCR was conductedusing the same components and conditions as described above foramplification of the YAT1::Yl Ale1 fusion fragment, prior to synthesisof pY168.

The PCR product comprising YlLPAAT1 ORF was digested with PciI and NotIand then utilized in a 3-way ligation with two fragments from pY168.Specifically, the YlLPAAT1 fragment was ligated with a 3530 bp Sph-Not1pY168 fragment and a 4248 bp NcoI-SphI pY168 fragment, to produce pY207,comprising a chimeric YAT1::YlLPAAT1::Lip1 gene. The Y. lipolyticaLPAAT1 ORF was confirmed by DNA sequencing. The components present inpY207 (FIG. 11B; SEQ ID NO:87) are identical to those present in pY201,with the exception of the chimeric YAT1::Yl LPAAT1::Lip1 gene in pY207,instead of the YAT1::ScAle1S::Lip1 gene in pY201 (FIG. 10A). Note thatYlLPAAT1 is labeled as “Yl LPAT1 ORF” in FIG. 11B.

Construction of pY175, Comprising a Caenorhabditis elegans LPCAT Gene

The Caenorhabditis elegans ORF designated as “CeLPCAT” (SEQ ID NO:1) wasoptimized for expression in Yarrowia lipolytica, by GenScriptCorporation (Piscataway, N.J.). In addition to codon optimization, 5′Nco1 and 3′ Not1 cloning sites were introduced within the synthetic gene(i.e., CeLPCATS; SEQ ID NO:6). None of the modifications in the CeLPCATSgene changed the amino acid sequence of the encoded protein (i.e., theprotein sequence encoded by the codon-optimized gene [i.e., SEQ ID NO:7]is identical to that of the wildtype protein sequence [i.e., SEQ IDNO:2]).

A Nco1-Not1 fragment comprising CeLPCATS was used to create pY175 (SEQID NO:88), in a 3-way ligation with two fragments from pY168 (SEQ IDNO:83). Specifically, the Nco1-Not1 fragment comprising CeLPCATS wasligated with a 3530 bp Sph-NotI pY168 fragment and a 4248 bp NcoI-SphIpY168 fragment to result in pY175. The components present in pY175 (FIG.12A; SEQ ID NO:88) are identical to those present in pY201, with theexception of the YAT1::CeLPCATS::Lip1 gene in pY175, instead of theYAT1::ScAle1S::Lip1 gene in pY201 (FIG. 10A). Note that CeLPCATS islabeled as “Ce.LPCATsyn” in FIG. 12A.

Construction of pY153, Comprising a Caenorhabditis elegans LPCAT Gene

The NcoI-NotI fragment comprising CeLPCATS, supra, was used to createpY153 (SEQ ID NO:89; FIG. 12B). In addition to comprising a chimericFBAIN::CeLPCATS::3′ Yl LPAAT1 gene, pY153 also contains a Yarrowialipolytica URA3 selection marker. Both the chimeric FBAIN::CeLPCATS::3′Yl LPAAT1 gene and the URA3 gene were flanked by fragments havinghomology to 5′ and 3′ regions of the Yarrowia lipolytica LPAAT1 gene tofacilitate integration by double homologous recombination, althoughintegration into Yarrowia lipolytica is known to usually occur withouthomologous recombination. Thus, construct pY153 thereby contained thefollowing components:

TABLE 16 Description of Plasmid pY153 (SEQ ID NO: 89) RE Sites AndNucleotides Within Description Of Fragment And Chimeric Gene SEQ ID NO:89 Components Cla1/Sap1 5′ portion of Yarrowia lipolytica gene encodingLPAAT1 (1-1398 bp) (GenBank Accession No. XP_504127) (i.e., bp 1-1112[complementary] in pY153); Sap1/Xba1 Vector backbone including:(1398-3993 bp) ColE1 plasmid origin of replication (i.e., bp 1380-2260in pY153); Ampicillin-resistance gene (Amp^(R)) for selection in E. coli(i.e., bp 2330-3190 [complementary] in pY153); E. coli f1 origin ofreplication (i.e., bp 3370-3770 in pY153) Xba1/Pme1 FBAIN::CeLPCATS::3′Yl LPAAT1, comprising: (3993-6719 bp) FBAINm: Yarrowia lipolytica FBAINpromoter (U.S. [a Nco1 site, located Pat. No. 7,202,356) (i.e., bp5756-6719 between CeLPCATS [complementary] in pY153); and FBAIN isCeLPCATS: codon-optimized LPCAT (SEQ ID present at bp 5756; NO: 6)derived from Caenorhabditis elegans T06E8.1 a Not1 site, located(GenBank Accession No. CAA98276) (i.e., bp 4910-5758 between CeLPCATS[complementary] in pY153; labeled as and YlLPAAT1 is “Ce.LPCATsyn” inFigure); present at bp 4904] 3′ Yl LPAAT1: 3′ untranslated portion ofYarrowia lipolytica gene encoding LPAAT1 (GenBank Accession No.XP_504127) (i.e., bp 3987-4905 [complementary] in pY153) Pme1-ClaIYarrowia lipolytica URA3 gene (GenBank Accession (6719-1 bp) No.AJ306421) (i.e., bp 6729-1 [complementary] in pY153)

Example 4 Functional Characterization of Different LPLATs inEPA-Producing Yarrowia libolytica Strain Y8406

Yarrowia lipolytica strain Y8406U, producing EPA, was used tofunctionally characterize the effects of overexpression of theSaccharomyces cerevisiae Ale1, Yarrowia lipolytica Ale1, Mortierellaalpina LPAAT1, Yarrowia lipolytica LPAAT1 and Caenorhabditis elegansLPCAT, following their stable integration into the Yarrowia hostchromosome. This was in spite of the host containing its native LPLATs,i.e., Ale1 and LPAAT1.

Transformation and Growth

Yarrowia lipolytica strain Y8406U (Example 1) was individuallytransformed with linear SphI-AscI fragments of the integrating vectorsdescribed in Example 3, wherein each LPLAT was under the control of theYarrowia YAT promoter. Specifically, vectors pY201(YAT1::ScAle1S::Lip1), pY168 (YAT1::YlAle1::Lip1), pY208(YAT1::MaLPAAT1S::Lip1), pY207 (YAT1::YlLPAAT1::Lip1) and pY175(YAT1::CeLPCATS::Lip1) were transformed according to the GeneralMethods.

Each transformation mix was plated on MM agar plates. Several resultantURA+ transformants were picked and inoculated into 3 mL FM medium(Biomyx Cat. No. CM-6681, Biomyx Technology, San Diego, Calif.)containing per L: 6.7 g Difco Yeast Nitrogen Base without amino acids, 5g Yeast Extract, 6 g KH₂PO₄, 2 g K₂HPO₄, 1.5 g MgSO₄.7H₂0, 1.5 mgthiamine.HCl, and 20 g glucose. After 2 days growth on a shaker at 200rpm and 30° C., the cultures were harvested by centrifugation andresuspended in 3 mL HGM medium (Cat. No. 2G2080, Teknova Inc.,Hollister, Calif.) containing 0.63% monopotassium phosphate, 2.7%dipotassium phosphate, 8.0% glucose, adjusted to pH 7.5. After 5 daysgrowth on a shaker at 200 rpm and at 30° C., 1 mL aliquots of thecultures were harvested by centrifugation and analyzed by GC.Specifically, the cultured cells were collected by centrifugation for 1min at 13,000 rpm, total lipids were extracted, and fatty acid methylesters [“FAMEs”] were prepared by trans-esterification, and subsequentlyanalyzed with a Hewlett-Packard 6890 GC (General Methods).

Based on the fatty acid composition of the 3 mL cultures, selectedtransformants were further characterized by flask assay. Specifically,clones #5 and #11 of strain Y8406U transformed with expression vectorpY201 (comprising ScAle1S) were selected and designated as“Y8406U::ScAle1S-5” and “Y8406U::ScAle1S-11”, respectively; clone #16 ofstrain Y8406U transformed with expression vector pY168 (comprisingYlAle1) was selected and designated as “Y8406U::YlAle1”; clone #8 ofstrain Y8406U transformed with expression vector pY208 (comprisingMaLPAAT1S) was selected and designated as “Y8406U::MaLPAAT1S”; clone #21of strain Y8406U transformed with expression vector pY207 (comprisingYlLPAAT1) was selected and designated as “Y8406U::YlLPAAT1”; and clone#23 of strain Y8406U transformed with expression vector pY175(comprising CeLPCATS) was selected and designated as “Y8406U::CeLPCATS”.Additionally, strain Y8406 (a Ura+ strain that was parent to strainY8406U (Ura−)) was used as a control.

Each selected transformant and the control was streaked onto MM agarplates. Then, one loop of freshly streaked cells was inoculated into 3mL FM medium and grown overnight at 250 rpm and 30° C. The OD_(600nm)was measured and an aliquot of the cells were added to a finalOD_(600nm) of 0.3 in 25 mL FM medium in a 125 mL flask. After 2 days ina shaker incubator at 250 rpm and at 30° C., 6 mL of the culture washarvested by centrifugation and resuspended in 25 mL HGM in a 125 mLflask. After 5 days in a shaker incubator at 250 rpm and at 30° C., a 1mL aliquot was used for GC analysis (supra) and 10 mL dried for dry cellweight [“DCW”] determination.

For DCW determination, 10 mL culture was harvested by centrifugation for5 min at 4000 rpm in a Beckman GH-3.8 rotor in a Beckman GS-6Rcentrifuge. The pellet was resuspended in 25 mL of water andre-harvested as above. The washed pellet was re-suspended in 20 mL ofwater and transferred to a pre-weighed aluminum pan. The cell suspensionwas dried overnight in a vacuum oven at 80° C. The weight of the cellswas determined.

Lipid Content, Fatty Acid Composition and Conversion Efficiencies

A total of four separate experiments were conducted under identicalconditions. Experiment 1 compared control strain Y8406 versus strainY8406U::ScAle1S-5. Experiment 2 compared control strain Y8406 versusstrain Y8406U::YlAle1. Experiment 3 compared control strain Y8406 versusstrain Y8406U::YlAle1, strain Y8406U::ScAle1S-11, and strainY8406U::MaLPAAT1S. Experiment 4 compared control strain Y8406 versusstrain Y8406U::MaLPAAT1S, strain Y8406U::YlLPAAT1 and strainY8406U::CeLPCATS.

In each experiment, the lipid content, fatty acid composition and EPA asa percent of the DCW are quantified for 1, 2 or 3 replicate cultures[“Replicates”] of the control Y8406 strain and the transformant Y8406Ustrain(s). Additionally, data for each Y8406U transformant is presentedas a % of the Y8406 control. Table 17 below summarizes the total lipidcontent of cells [“TFAs % DCW”], the concentration of each fatty acid asa weight percent of TFAs [“% TFAs”] and the EPA content as a percent ofthe dry cell weight [“EPA % DCW”]. More specifically, fatty acids areidentified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0 (stearicacid), 18:1 (oleic acid), 18:2 (LA), ALA, EDA, DGLA, ARA, ETrA, ETA andEPA.

Table 18 summarizes the conversion efficiency of each desaturase and theΔ9 elongase functioning in the PUFA biosynthetic pathway and which arerequired for EPA production. Specifically, the Δ2 desaturase conversionefficiency [“Δ12 CE”], Δ8 desaturase conversion efficiency [“Δ8 CE”], Δ5desaturase conversion efficiency [“Δ5 CE”], Δ17 desaturase conversionefficiency [“Δ17 CE”] and Δ9 elongation conversion efficiency [“Δ9e CE”]are provided for each control Y8406 strain and the transformant Y8406Ustrain(s); data for each Y8406U transformant is presented as a % of theY8406 control. Conversion efficiency was calculated according to theformula: product(s)/(product(s)+substrate)*100, where product includesboth product and product derivatives.

TABLE 17 Lipid Content And Composition In LPCAT Transformant Strains OfYarrowia lipolytica Y8406 TFA % TFAs EPA Expt. Strain Replicates % DCW16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA ERA ETA EPA % DCW 1 Y8406AVG.3 17.6 3.8 0.7 3.3 6.4 22.6 2.5 2.8 2.2 0.5 1.9 2.0 48.9 8.6Y8406U:: AVG.3 18.3 4.2 0.7 3.5 5.7 15.1 0.6 3.3 3.7 0.8 1.8 2.3 56.910.4 ScAle1S-5 % Ctrl 104 111 100 106 89 67 24 118 168 160 95 115 116121 2 Y8406 AVG.3 23.2 3.5 0.6 3.3 6.4 22.3 2.7 2.6 2.1 0.5 1.6 2.0 49.911.6 Y8406U:: AVG.3 22.3 3.8 0.7 2.9 3.9 12.7 0.4 3.0 3.8 0.8 1.6 2.460.9 13.6 YlAle1 % Ctrl 96 109 117 88 61 57 15 115 181 160 100 120 122117 3 Y8406 1 26.1 2.7 0.7 2.8 6.5 20.5 2.5 3.2 2.3 0.7 0.8 0.0 50.813.3 Y8406U:: AVG.2 23.3 3.3 0.7 2.4 3.6 12.1 0.5 3.2 3.5 0.9 0.0 2.362.2 14.5 YlAle1 % Ctrl 89 122 100 86 55 59 20 100 152 129 0 na 122 109Y8406U:: AVG.2 28.0 3.0 0.7 3.0 5.5 13.1 0.6 3.5 3.8 0.9 0.0 2.4 58.516.4 ScAle1S-11 % Ctrl 107 111 100 107 85 64 24 109 165 129 0 na 115 123Y8406U:: AVG.2 23.7 4.4 0.8 4.2 6.6 11.2 0.7 2.7 3.7 0.9 0.0 2.5 57.013.5 MaLPAAT1S % Ctrl 91 163 114 150 102 55 28 84 161 129 0 na 112 102 4Y8406 AVG.2 27.9 2.8 0.6 3.1 6.2 20.6 2.9 2.9 2.0 0.6 0.7 2.0 49.4 13.8Y8406U:: AVG.2 25.2 4.8 0.8 4.8 6.9 11.6 0.8 2.5 3.0 0.7 0.0 2.3 55.314.0 MaLPAAT1S % Ctrl 90 171 133 155 111 56 28 86 150 117 0 115 112 101Y8406U:: AVG.2 25.2 3.7 0.7 4.2 6.2 13.0 1.2 2.3 2.6 0.6 0.0 2.2 56.714.3 YlLPAAT1 % Ctrl 90 132 117 135 100 63 41 79 130 100 0 110 115 104Y8406U:: AVG.2 24.7 3.8 0.6 4.6 7.1 13.9 1.6 2.3 2.6 0.6 0.4 2.2 53.613.2 CeLPCATS % Ctrl 89 136 100 148 115 67 55 79 130 100 57 110 109 96

TABLE 18 Desaturase And Elongase Conversion Efficiency In LPCATTransformant Strains Of Yarrowia lipolytica Y8406 Expt. StrainReplicates Δ12 CE Δ9e CE Δ8 CE Δ5 CE Δ17 CE 1 Y8406 AVG.3 93 70 92 92 90Y8406U::ScAle1S-5 AVG.3 94 81 93 91 89 % Ctrl 101 116 101 98 98 2 Y8406AVG.3 93 70 93 93 91 Y8406U::YlAle1 AVG.3 96 85 94 91 90 % Ctrl 103 121101 98 98 3 Y8406 1 93 72 93 96 89 Y8406U::YlAle1 AVG.2 96 85 96 92 89 %Ctrl 104 119 103 96 100 Y8406U::ScAle1S-11 AVG.2 94 83 95 91 88 % Ctrl101 117 102 95 99 Y8406U::MaLPAAT1S AVG.2 92 85 96 90 89 % Ctrl 100 119103 94 100 4 Y8406 AVG.2 93 71 94 93 91 Y8406U::MaLPAAT1S AVG.2 92 84 9691 90 % Ctrl 99 118 102 99 100 Y8406U::YlLPAAT1 AVG.2 93 82 96 92 92 %Ctrl 100 115 103 100 101 Y8406U::CeLPCATS AVG.2 92 80 96 92 91 % Ctrl 99113 102 99 100

Based on the data concerning Experiments 1, 2 and 3 in Table 17 andTable 18, overexpression of LPLAT in EPA strains Y8406U::ScAle1S-5,Y8406U::ScAle1S-11, Y8406U::YlAle1 and Y8406U::MaLPAAT1S results insignificant reduction (to 67% or below of the control) of theconcentration of LA (18:2) as a weight % of TFAs [“LA % TFAs”], anincrease (to at least 12% of the control) in the concentration of EPA asa weight % of TFAs [“EPA % TFAs”], and an increase (to at least 16% ofthe control) in the conversion efficiency of the Δ9 elongase. Comparedto Y8406U::ScAle1S-5 and Y8406U::ScAle1S-11, Y8406U::YlAle1 has lower LA% TFAs, higher EPA % TFAs, better Δ9 elongation conversion efficiency,and slightly lower TFAs % DCW and EPA % DCW. Y8406U::Yl Ale1 andY8406U::MaLPAAT1S are similar except overexpression of MaLPAAT1Sresulted in lower LA % TFAs, EPA % TFAs, and EPA % DCW.

Experiment 4 shows that overexpression of LPLAT in EPA strainsY8406U::YlLPAAT1, Y8406U::MaLPAAT1S and Y8406U::CeLPCATS results insignificant reduction (to 67% or below of the control) of LA % TFAs, anincrease (to at least 9% of the control) in EPA % TFAs, and an increase(to at least 13% of the control) in the conversion efficiency of the Δ9elongase. Compared to Y8406U::CeLPCATS, Y8406U::YlLPAAT1 andY8406U::MaLPAAT1S both have lower LA % TFAs, higher EPA % TFAs, higherEPA % DCW, and slightly better TFAs % DCW. Y8406U::YlLPAAT1 andY8406U::MaLPAAT1S are similar except overexpression of MaLPAAT1S resultsin lower LA % TFAs, slightly lower EPA % TFAs and EPA % DCW, andslightly better Δ9 elongase conversion efficiency.

It is well known in the art that most desaturations occur at the sn-2position of phospholipids, while fatty acid elongations occur onacyl-CoAs. Furthermore, ScAle1S, YlAle1, MaLPAAT1S and YlLPAAT1 wereexpected to only incorporate acyl groups from the acyl-CoA pool into thesn-2 position of lysophospholipids, such as lysophosphatidic acid[“LPA”] and lysophosphatidylcholine [“LPC”]. Thus, it was expected thatexpression of ScAle1S, YlAle1, MaLPAAT1S, and YlLPAAT1 would result inimproved desaturations due to improved substrate availability inphospholipids, and not result in improved elongations that requireimproved substrate availability in the CoA pool. Our data (supra) showsthat unexpectedly, expression of ScAle1S, YlAle1, MaLPAAT1S, andYlLPAAT1 significantly improved the Δ9 elongase conversion efficiency instrains of Yarrowia producing EPA but did not improve the desaturations(measured as Δ2 desaturase conversion efficiency, Δ8 desaturaseconversion efficiency, Δ5 desaturase conversion efficiency or Δ17desaturase conversion efficiency).

CeLPCAT was previously shown to improve Δ6 elongation conversionefficiency in Saccharomyces cerevisiae fed LA or GLA (Intl. App. Pub.No. WO 2004/076617). This was attributed to its reversible LPCATactivity that released fatty acids from phospholipids into the CoA pool.An improvement in Δ9 elongation conversion efficiency in an oleaginousmicrobe, such as Yarrowia lipolytica, engineered for high level LC-PUFAproduction in the absence of feeding fatty acids was not contemplated inIntl. App. Pub. No. WO 2004/076617.

Furthermore, expression of ScAle1S, YlAle1, MaLPAAT1S, YlLPAAT1 andCeLPCATS did not significantly alter either the level of PUFAsaccumulated or the total lipid content in strains of Yarrowia producingEPA.

Previous studies have shown that both Δ6 elongation and Δ9 elongationare bottlenecks in long chain PUFA biosynthesis due to poor transfer ofacyl groups between phospholipid and acyl-CoA pools. Based on theimproved Δ9 elongase conversion efficiency resulting fromover-expression of LPLATs, demonstrated above, it is anticipated thatthe LPLATs described herein and their orthologs, such as Sc LPAAT, willalso improve Δ6 elongation conversion efficiency.

Example 5 Functional Characterization of Different LPLATs inDHA-Producing Y. lipolytica Strain Y5037

Yarrowia lipolytica strain Y5037U, producing DHA, was used tofunctionally characterize the effects of overexpression of theSaccharomyces cerevisiae Ale1, Mortierella alpina LPAAT1 andCaenorhabditis elegans LPCAT, following their stable integration intothe Yarrowia host chromosome. This was in spite of the host containingits native LPLATs, i.e., Ale1 and LPAAT1.

Transformation and Growth

Yarrowia lipolytica strain Y5037U (Example 2) was individuallytransformed with linear SphI-AscI fragments of the integrating vectorsdescribed in Example 3, wherein ScAle1S and MaLPAAT1S were under thecontrol of the Yarrowia YAT promoter, while CeLPCATS was under thecontrol of the Yarrowia FBAIN promoter. Specifically, vectors pY201(YAT1::ScAle1S::Lip1), pY208 (YAT1::MaLPAAT1S::Lip1) and pY153(FBAIN::CeLPCATS::YlLPAAT1) were transformed according to the GeneralMethods.

Each transformation mix was plated on MM agar plates. Selectedtransformants were further characterized, as detailed below. Morespecifically, clone #7 of strain Y5037U, transformed with expressionvector pY153 (comprising CeLPCATS) was selected and designated as“Y5037U::FBAIN-CeLPCATS”; clone #18 of strain Y5037U, transformed withexpression vector pY201 (comprising ScAle1S) was selected and designatedas “Y5037U::ScAle1S”; and clone #6 of strain Y5037U, transformed withexpression vector pY208 (comprising MaLPAAT1S) was selected anddesignated as “Y5037U::MaLPAAT1S”. Additionally, strain Y5037 (a Ura+strain that was parent to strain Y5037 (Ura−)) was used as a control.

A total of four separate experiments were conducted in 3 mL culturebased on variable culturing conditions and strains, to examine theeffect of LPLAT overexpression on lipid content, fatty acid compositionand conversion efficiencies. Experiment 1 compared control strain Y5037versus strains Y5037U::FBAIN-CeLPCATS and Y5037U::ScAleIS after 2 daysof growth in MM medium on a shaker at 200 rpm and 30° C., followed by 3days of incubation in 3 mL HGM medium. MM medium (Cat. No. CML-MM,Biomyx Technology), pH 6.1, contains per L: 1.7 g yeast nitrogen base[“YNB”]without amino acids and NH₄SO₄, 1 g proline, 0.1 g adenine, 0.1 glysine, and 20 g glucose.

Experiment 2 compared control strain Y5037 versus strain Y5037U::ScAleISafter 2 days of growth in CSM-U medium on a shaker at 200 rpm and 30°C., followed by 3 days of incubation in 3 mL HGM medium. CSM-U medium(Cat. No C8140, Teknova Inc, Hollister, Calif.) contains: 0.13% aminoacid dropout powder minus uracil, 0.17% yeast nitrogen base, 0.5%(NH₄)₂SO₄, and 2.0% glucose.

Experiment 3 compared control strain Y5037 versus strainsY5037U::FBAIN-CeLPCATS and Y5037U::ScAleIS after 2 days of growth in MMmedium on a shaker at 200 rpm and 30° C., followed by 5 days ofincubation in 3 mL HGM medium.

Experiment 5 compared control strain Y5037 versus strainY5037U::MaLPAAT1S after 2 days of growth in FM medium on a shaker at 200rpm and 30° C., followed by 3 days of incubation in 3 mL HGM medium. Thecomposition of FM medium is described in Example 4.

Following growth for 3 days (Experiments 1, 2, and 5) or 5 days(Experiment 3) in HGM, 1 mL aliquots of the cultures were harvested bycentrifugation and analyzed by GC, as described in Example 4.

Experiment 4 compared control strain Y5037 versus strainsY5037U::FBAIN-CeLPCATS and Y5037U::ScAleIS after 2 days of growth in 25mL FM medium followed by 5 days of incubation in HGM medium as describedabove. Specifically, one loop of freshly streaked cells from MM agarplates was inoculated into 3 mL FM medium and grown overnight at 250 rpmand 30° C. The OD_(600nm) was measured and an aliquot of the cells wereadded to a final OD_(600nm) of 0.3 in 25 mL FM medium in a 125 mL flask.After 2 days in a shaker incubator at 250 rpm and at 30° C., 6 mL of theculture was harvested by centrifugation and resuspended in 25 mL HGM ina 125 mL flask. After 5 days in a shaker incubator at 250 rpm and at 30°C., a 1 mL aliquot was used for GC analysis and 10 mL dried for dry cellweight [“DCW”] determination (supra, Example 4).

Lipid Content, Fatty Acid Composition and Conversion Efficiencies

In each experiment, the lipid content and fatty acid composition arequantified for 1, 2, 3 or 4 replicate cultures [“Replicates”] of thecontrol Y5037 strain and the transformant Y5037U strain(s).Additionally, data for each Y5037U transformant is presented as a % ofthe Y5037 control. Table 19 below summarizes the concentration of eachfatty acid as a weight percent of TFAs [“% TFAs”]. More specifically,fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid),18:0 (stearic acid), 18:1 (oleic acid), 18:2 (LA), ALA, EDA, DGLA, ARA,ETrA, ETA, EPA, DPA, DHA and EDD (corresponding to the sum of EPA plusDPA plus DHA). Additionally, the ratio of DHA % TFAs/DPA % TFAs isprovided.

Table 20 summarizes the total DCW (mg/mL), the total lipid content ofcells [“TFAs % DCW”], and the conversion efficiency of each desaturaseand elongase functioning in the PUFA biosynthetic pathway and which arerequired for DHA production. Specifically, the Δ2 desaturase conversionefficiency [“Δ2 CE”], Δ8 desaturase conversion efficiency [“Δ8 CE”], Δ5desaturase conversion efficiency [“Δ5 CE”], Δ17 desaturase conversionefficiency [“Δ17 CE”], Δ4 desaturase conversion efficiency [“Δ4 CE”], Δ9elongation conversion efficiency [“Δ9e CE”] and Δ5 elongation conversionefficiency [“Δ5e CE”] are provided for each control Y5037 strain and thetransformant Y5037U strain(s); data for each Y5037U transformant ispresented as a % of the Y5037 control. Conversion efficiency wascalculated according to the formula:product(s)/(product(s)+substrate)*100, where product includes bothproduct and product derivatives.

TABLE 19 Lipid Content and Composition In LPCAT Transformant Strains OfYarrowia lipolytica Y5037 % TFAs Repli- DHA/ Expt. Strain cates 16:016:1 18:0 18:1 18:2 ALA EDA DGLA ARA ETrA ETA EPA DPA DHA EDD DPA 1Y5037 AVG.4 4.1 1.1 3.2 5.4 21.8 0.5 2.9 1.7 0.7 1.3 1.8 18.1 20.6 6.545.2 0.3 Y5037U:: 1 5.2 1.2 2.7 8.5 11.1 0.3 2.5 3.6 1.1 1.4 2.7 31.79.6 11.0 52.4 1.1 FBAIN- % Ctrl 127 109 84 157 51 60 86 212 157 108 150175 47 169 116 367 CeLPCATS Y5037U:: 1 4.4 1.4 2.0 4.4 15.7 0.5 3.5 2.71.0 1.2 2.2 22.0 16.8 14.4 53.3 0.9 ScAleIS % Ctrl 107 127 63 81 72 100121 159 143 92 122 122 82 222 118 300 2 Y5037 AVG.2 4.4 1.1 3.9 5.4 21.80.5 3.4 1.6 0.8 1.1 1.7 17.0 21.0 6.7 44.7 0.3 Y5037U:: 1 4.5 1.5 2.54.7 16.6 0.4 4.1 2.6 1.1 1.1 2.1 21.2 17.1 13.2 51.5 0.8 ScAleIS % Ctrl102 136 64 87 76 80 121 163 138 100 124 125 81 197 115 267 3 Y5037 AVG.33.9 1.1 1.6 4.7 20.7 0.5 3.3 1.8 1.3 1.5 3.9 19.3 20.8 7.9 47.9 0.4Y5037U:: 1 5.8 1.1 2.6 8.0 10.0 0.3 3.0 3.6 1.9 2.2 2.7 31.0 9.9 11.852.7 1.2 FBAIN- % Ctrl 149 100 163 170 48 60 91 200 146 147 69 161 48149 110 300 CeLPCATS Y5037U:: 1 4.6 1.3 1.8 5.9 18.1 0.3 4.4 2.4 1.3 1.84.0 22.1 15.1 11.7 48.9 0.8 ScAleIS % Ctrl 118 118 113 126 87 60 133 133100 120 103 115 73 148 102 200 5 Y5037 1 5.1 1.3 1.6 4.7 22.5 2.7 3.91.9 1.4 1.3 1.7 20.4 20.7 8.9 50.1 0.4 Y5037U:: 1 6.1 1.5 1.8 4.5 21.12.2 4.0 2.1 1.5 1.2 1.7 23.4 19.5 10.7 53.7 0.6 MaLPAT1 % Ctrl 120 115113 96 94 81 103 111 107 92 100 115 94 120 107 150 4 Y5037 AVG.3 3.9 1.21.3 5.9 22.4 3.9 1.7 1.8 0.8 1.0 1.6 20.0 26.2 6.7 52.9 0.3 Y5037U::AVG.3 6.1 1.3 3.4 8.8 10.1 0.7 1.6 3.5 0.7 1.3 2.3 33.9 12.5 10.6 57.00.9 FBAIN- % Ctrl 156 108 262 149 45 18 94 194 88 130 144 170 48 158 108300 CeLPCATS Y5037U:: AVG.3 5.4 1.4 2.7 8.7 21.1 1.7 5.4 2.5 0.6 1.2 1.420.4 19.6 7.3 47.3 0.4 ScAleIS % Ctrl 138 117 208 147 94 44 318 139 75120 88 102 75 109 89 133

TABLE 20 Desaturase And Elongase Conversion Efficiency In LPCATTransformant Strains Of Yarrowia lipolytica Y5037 DCW TFA % Δ12 Δ9e Δ17Δ5e Expt. Strain Replicates mg/mL DCW CE CE Δ8 CE Δ5 CE CE CE Δ4 CE 1Y5037 AVG.4 nd nd 93 71 92 93 90 60 24 Y5037U:: 1 nd nd 90 85 94 89 8939 53 FBAIN- % Ctrl nd nd 96 120 102 96 98 66 221 CeLPCATS Y5037U:: 1 ndnd 95 80 93 92 89 59 46 ScAleIS % Ctrl nd nd 102 113 101 99 98 98 191 2Y5037 AVG.2 nd nd 93 71 91 93 89 62 24 Y5037U:: 1 nd nd 94 79 92 92 8859 44 ScAleIS % Ctrl nd nd 101 111 100 99 98 95 180 3 Y5037 AVG.3 nd nd94 74 92 90 89 60 27 Y5037U:: 1 nd nd 91 86 92 90 87 41 54 FBAIN- % Ctrlnd nd 96 117 100 100 97 69 198 CeLPCATS Y5037U:: 1 nd nd 93 77 90 89 8755 44 ScAleIS % Ctrl nd nd 99 105 98 99 97 92 160 5 Y5037 1 nd nd 95 7091 93 88 59 30 Y5037U:: 1 nd nd 95 73 92 93 88 56 36 MaLPAT1 % Ctrl ndnd 100 104 101 100 100 95 118 4 Y5037 AVG.3 3.7 19.7 nd 69 95 94 93 6220 Y5037U:: AVG.3 3.0 14.0 nd 86 96 91 91 40 46 FBAIN- % Ctrl 82 71 nd124 100 97 98 65 226 CeLPCATS Y5037U:: AVG.3 3.7 31.6 nd 72 89 92 85 5727 ScAleIS % Ctrl 101 157 nd 104 93 98 92 92 133

Based on the data in Table 19 and Table 20, overexpression of LPLAT inDHA strains Y5037U::CeLPCATS, Y5037U::ScAleIS and Y5037U::MaLPAAT1Sresults in reduction of the concentration of LA as a weight % of TFAs[“LA % TFAs”], an increase in the concentration of EPA as a weight % ofTFAs [“EPA % TFAs”], an increase in the concentration of DHA as a weight% of TFAs [“DHA % TFAs”], an increase in the concentration ofEPA+DPA+DHA as a weight % of TFAs [“EDD % TFAs”] (with the exception ofstrain Y5037U::ScAleIS in Experiment 4), an increase in the ratio of DHA% TFAs to DPA % TFAs [“DHA/DPA”], an increase in the conversionefficiency of the Δ9 elongase and an increase in the conversionefficiency of the Δ4 desaturase.

More specifically, depending on the culture conditions, CeLPCATSoverexpression in Y5037U::CeLPCATS can reduce LA % TFAs to 45%, increaseEPA % TFAs to 175%, increase DHA % TFAs to 169%, increase Δ9 elongationCE to 124%, and increase Δ4 desaturation CE to 226%, as compared to thecontrol. Similarly, depending on the culture conditions, ScAle1Soverexpression in Y5037U::ScAleIS can reduce LA % TFAs to 72%, increaseEPA % TFAs to 125%, increase DHA % TFAs to 222%, increase Δ9 elongationCE to 113%, and increase Δ4 desaturation CE to 191%, as compared to thecontrol. Finally, overexpression of MaLPAAT1 in Y5037U::MaLPAAT1S canreduce LA % TFAs to 94%, increase EPA % TFAs to 115%, increase DHA %TFAs to 120%, increase Δ9 elongation CE to 104%, and increase Δ4desaturation CE to 118%, as compared to the control.

Although Y5037U::CeLPCATS possessed a significantly lower total lipidcontent [“TFAs % DCW”] in Experiment 4, the total lipid content wassignificantly improved in strain Y5037U::ScAleIS. This increase in lipidcontent is a likely explanation for the lower EDD % TFAs in strainY5037U::ScAleIS.

DHA biosynthesis via EPA involves two steps: elongation of EPA to DPA byC_(20/22) elongase (also known as either a “C20” elongase or a Δ5elongase) and desaturation of DPA to DHA by Δ4 desaturase. An importantbottleneck in the production of DHA from EPA has been the Δ4desaturation step, evident by the build up of DPA, although themechanistic details for this limitation were unknown. The results aboveshow that expression of ScAle1S, YlAle1, YlLPAAT1, MaLPAAT1S, andCeLPCATS proteins significantly improved Δ4 desaturation. Thus, Δ4desaturation was not limiting because of Δ4 desaturase activity per se.Instead, Δ4 desaturation was limiting because of limited availability ofthe DPA substrate at the sn-2 position of phospholipids. The resultsshowed unexpectedly that (unlike other desaturation substrates), limitedDPA incorporation into phospholipid can be overcome by overexpression ofAle1, LPAAT and LPCAT proteins.

Previously, Intl. App. Pub. No. WO 2004/076617 showed that expression ofCeLPCAT (SEQ ID NO:2) in Saccharomyces cerevisiae improved Δ6 elongationof exogenously provided GLA to DGLA. It hypothesized that CeLPCATremoved an acyl chain from the sn-2 position of phospholipids, therebymaking the removed acyl group available for elongation in the CoA pool.It was shown in the present studies that the expression of thecodon-optimized CeLPCATS, under control of the YAT1 promoter, in strainsof Yarrowia lipolytica engineered to produce high levels of EPA (Example4) and DHA (Example 5), respectively, improves Δ9 elongation ofendogenously produced LA to EDA. However, expression of CeLPCATS inDHA-producing strain Y5037U::CeLPCATS unexpectedly did not result inimproved Δ5 elongation of EPA to DPA. In contrast, expression ofCeLPCATS in DHA-producing strain Y5037U::CeLPCATS very significantlyimproved Δ4 desaturation of DPA to DHA (supra). This is especiallyunexpected since desaturations occur mainly at the sn-2 position ofphospholipids and elongation occurs in the CoA pool.

Based on the improved Δ4 desaturation conversion efficiency resultingfrom over-expression of LPLATs, demonstrated above, it is anticipatedthat the LPLATs described herein and their orthologs, such as ScLPAAT,will also improve Δ4 desaturation conversion efficiency.

Example 6 Functional Characterization of Different LPLATs inARA-Producing Y. lipolytica Strain Y8006U

Yarrowia lipolytica strain Y8006U, producing ARA, is used tofunctionally characterize the effects of overexpression of theSaccharomyces cerevisiae Ale1, Mortierella alpina LPAAT1 andCaenorhabditis elegans LPCAT, following their integration into theYarrowia host chromosome. This was in spite of the host containing itsnative LPLATs, i.e., Ale1 and LPAAT1.

Transformation and Growth

Yarrowia lipolytica strain Y8006U (Example 1) will be individuallytransformed with linear SphI-AscI fragments of the integrating vectorsdescribed in Example 3, in a manner comparable to that utilized inExample 4. URA+ transformants will be selected, grown for 2 days in FMmedium and 5 days in HGM medium and then 1 mL aliquots of the cultureswill be harvested by centrifugation and analyzed by GC (Example 4).Based on the fatty acid composition of the 3 mL cultures, selectedtransformants will be further characterized using strain Y8006 (a Ura+strain that was parent to strain Y8006U (Ura−)) as a control.

Each selected transformant and the control will be re-grown in FM andHGM medium, as described in Example 4, and then subjected to GC analysisand DCW determination.

The lipid content, fatty acid composition and ARA as a percent of theDCW will be quantified for the control Y8006 strain and the transformantY8006U strain(s). Additionally, data for each Y8006U transformant willbe determined as a % of the Y8006 control. The conversion efficiency ofeach desaturase and the Δ9 elongase functioning in the PUFA biosyntheticpathway and which is required for ARA production will also be determinedand compared to the control, in a manner similar to that in Examples 4and 5.

It is hypothesized that overexpression of the ScAle1S, YlAle1,MaLPAAT1S, YlLPAAT1 and CeLPCATS LPLATs in the ARA strains will resultin a reduction of the concentration of LA (18:2) as a weight % of TFAs[“LA % TFAs”], an increase in the concentration of ARA as a weight % ofTFAs [“ARA % TFAs”], and an increase in the conversion efficiency of theΔ9 elongase.

Example 7 Construction of Expression Vectors Comprising LPAAT ORFs andan Autonomously Replicating Sequence

The present example describes the construction of vectors comprisingautonomously replicating sequences [“ARS”] and LPAAT ORFs suitable forLPAAT gene expression without integration in Yarrowia lipolytica. ORFsincluded the Saccharomyces cerevisiae LPAAT encoding SEQ ID NO:18 andthe Yarrowia lipolytica LPAAT1 encoding SEQ ID NO:17. Example 8describes the results obtained following transformation of these vectorsinto Y. lipolytica.

Construction of pY222, Comprising a Codon-Optimized Saccharomycescerevisiae LPAAT Gene

The Saccharomyces cerevisiae ORF designated as “ScLPAAT” (SEQ ID NO:18)was optimized for expression in Yarrowia lipolytica, by DNA 2.0 (MenloPark, Calif.). In addition to codon optimization, 5′ Pci1 and 3′ Not1cloning sites were introduced within the synthetic gene (i.e., ScLPAATS;SEQ ID NO:96). None of the modifications in the ScLPAATS gene changedthe amino acid sequence of the encoded protein (i.e., the proteinsequence encoded by the codon-optimized gene [i.e., SEQ ID NO:97] isidentical to that of the wildtype protein sequence [i.e., SEQ IDNO:18]). ScLPAATS was cloned into pJ201 (DNA 2.0) to result inpJ201:ScLPAATS.

A 926 bp Pci1/Not1 fragment comprising ScLPAATS was excised frompJ201:ScLPAATS and cloned into NcoI-Not1 cut pYAT-DG2-1 to create pY222(SEQ ID NO:100; Table 21; FIG. 13A). Thus, pY222 contained the followingcomponents:

TABLE 21 Description of Plasmid pY222 (SEQ ID NO: 100) RE Sites AndNucleotides Within Description Of Fragment SEQ ID NO: 100 And ChimericGene Components Sal1/SwaI YAT1::ScLPAATS::Lip1, comprising: (1-2032)YAT1: Yarrowia lipolytica YAT1 promoter (U.S. patent application Pub.No. 2006/0094102-A1); ScLPAATS: codon-optimized ScLPAATS (SEQ ID NO: 96)(labeled as “Sc LPAATs ORF” in Figure); Lip1: Lip1 terminator sequencefrom Yarrowia Lip1 gene (GenBank Accession No. Z50020) (labeled as“Lip1-3′” in Figure) SwaI/AvaI ColE1 plasmid origin of replication;(2032-4946) Ampicillin-resistance gene (Amp^(R)) for selection in E.coli; E. coli f1 origin of replication AvaI-SphI Yarrowia lipolyticacentromere and autonomously replicating (4946-6330) sequence [“ARS”] 18locus SphI-SalI Yarrowia lipolytica URA3 gene (GenBank Accession No.(6330-1) AJ306421)Construction of pY177, Comprising a Yarrowia lipolytica LPAAT1 Gene

The Yarrowia lipolytica centromere and autonomously replicating sequence[“ARS”] was amplified by standard PCR using primer 869 (SEQ ID NO:98)and primer 870 (SEQ ID NO:99), with plasmid pYAT-DG2-1 as template. ThePCR product was digested with AscI/AvrII and cloned into AscI-AvrIIdigested pY207 (SEQ ID NO:87; Example 3) to create pY177 (SEQ ID NO:101;Table 22; FIG. 13B). Thus, the components present in pY177 are identicalto those in pY207 (FIG. 11B), except for the replacement of the 373 bppY207 sequence between AscI and AvrII with the 1341 bp sequencecontaining ARS. More specifically, pY177 contained the followingcomponents:

TABLE 22 Description of Plasmid pY177 (SEQ ID NO: 101) RE Sites AndNucleotides Within Description Of Fragment And SEQ ID NO: 101 ChimericGene Components BsiW1/Sbf1 LoxP::Ura3::LoxP, comprising: (1-1706 bp)LoxP sequence (SEQ ID NO: 78) Yarrowia lipolytica Ura3 gene (GenBankAccession No. AJ306421); LoxP sequence (SEQ ID NO: 78) Sbf1/Sph1 3′portion of Yarrowia lipolytica POX3 Acyl-CoA oxidase 3 (1706-3043 bp)(GenBank Accession No. YALI0D24750g) SphI/AscI ColE1 plasmid origin ofreplication; (3043-5743 bp) Ampicillin-resistance gene (Amp^(R)) forselection in E. coli; E. coli f1 origin of replication AscI/BsiWI 5′portion of Yarrowia lipolytica POX3 Acyl-CoA oxidase 3 (5743-6513 bp)(GenBank Accession No. YALI0D24750g) AscI/AvrII Yarrowia lipolyticacentromere and autonomously replicating (5743-7084 bp) sequence [“ARS”]18 locus AvrII/BsiWI 5′ portion of Yarrowia lipolytica POX3 Acyl-CoAoxidase 3 (7084-7481 bp) (GenBank Accession No. YALI0D24750g)BsiWI/BsiWI YAT1::YlLPAAT1::Lip1, comprising: (7481-1 bp) YAT1: Yarrowialipolytica YAT1 promoter (U.S. patent application Pub. No.2006/0094102-A1); YlLPAAT1: Yarrowia lipolytica LPAAT1 (“YALI0E18964g”;GenBank Accession No. XP_504127) (SEQ ID NO: 16) (labeled as “Yl LPAT1ORF” in Figure); Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene(GenBank Accession No. Z50020) (labeled as “Lip1-3′” in Figure)

Example 8 Functional Characterization of Different LPAATs inEPA-Producing Yarrowia lipolytica Strain Y8406

Yarrowia lipolytica strain Y8406U, producing EPA, was used tofunctionally characterize the effects of expression of the Saccharomycescerevisiae LPAATS (SEQ ID NO:96) and Yarrowia lipolytica LPAAT1 (SEQ IDNO:16) without integration on self-replicating plasmids. This was inspite of the host containing its native LPAATs.

Transformation and Growth

Yarrowia lipolytica strain Y8406U (Example 1) was individuallytransformed with uncut plasmids from Example 7. Specifically, vectorspY177 (YAT1::YlLPAAT1::Lip1) [SEQ ID NO:101] and pY222(YAT1::ScLPAATS::Lip1) [SEQ ID NO:100] were transformed according to theGeneral Methods.

Each transformation mix was plated on MM agar plates. Several resultantURA+ transformants were picked and inoculated into 3 mL CSM-U medium(Teknova Cat. No. C8140, Teknova Inc., Hollister, Calif.), wherein CSM-Umedium refers to CM Broth with glucose minus uracil containing 0.13%amino acid dropout powder minus uracil, 0.17% yeast nitrogen base, 0.5%(NH₄)₂SO₄, and 2.0% glucose. After 2 days growth on a shaker at 200 rpmand 30° C., the cultures were harvested by centrifugation andresuspended in 3 mL HGM medium (Cat. No. 2G2080, Teknova Inc.). After 5days growth on a shaker, 1 mL aliquots of the cultures were harvestedand analyzed by GC, as described in Example 4.

Based on the fatty acid composition of the 3 mL cultures, selectedtransformants were further characterized by flask assay. Specifically,clones #5 and #6 of strain Y8406U transformed with expression vectorpY222 (comprising ScLPAATS) were selected and designated as“Y8406U::ScLPAATS-5” and “Y8406U::ScLPAATS-6”, respectively; clone #1 ofstrain Y8406U transformed with expression vector pY177 (comprisingYlLPAAT1) was selected and designated as “Y8406U::YlLPAAT1”.Additionally, strain Y8406 (a Ura+ strain that was parent to strainY8406U (Ura−)) was used as a control.

Each selected transformant and the control was streaked onto MM agarplates. Then, one loop of freshly streaked cells was inoculated into 3mL CSM-U medium and grown overnight at 250 rpm and 30° C. The OD_(600nm)was measured and an aliquot of the cells were added to a finalOD_(600nm) of 0.3 in 25 mL CSM-U medium in a 125 mL flask. After 2 daysin a shaker incubator at 250 rpm and at 30° C., 6 mL of the culture washarvested by centrifugation and resuspended in 25 mL HGM in a 125 mLflask. After 5 days in a shaker incubator at 250 rpm and at 30° C., a 1mL aliquot was used for GC analysis and 10 mL dried for dry cell weight[“DCW”] determination, as described in Example 4.

Lipid Content, Fatty Acid Composition and Conversion Efficiencies

The lipid content, fatty acid composition and EPA as a percent of theDCW are quantified for 2 replicate cultures [“Replicates”] of thecontrol Y8406 strain and the transformant Y8406U strain(s).Additionally, data for each Y8406U transformant is presented as a % ofthe Y8406 control. Table 23 below summarizes the total lipid content ofcells [“TFAs % DCW”], the concentration of each fatty acid as a weightpercent of TFAs [“% TFAs”] and the EPA content as a percent of the drycell weight [“EPA % DCW”]. More specifically, fatty acids are identifiedas 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0 (stearic acid), 18:1(oleic acid), 18:2 (LA), ALA, EDA, DGLA, ARA, ETrA, ETA and EPA.

Table 24 summarizes the conversion efficiency of each desaturase and theΔ9 elongase functioning in the PUFA biosynthetic pathway and which arerequired for EPA production, in a manner identical to that described inExample 4.

TABLE 23 Lipid Content And Composition In ScLPAATS and YlLPAAT1Transformant Strains Of Yarrowia lipolytica Y8406 TFA % % TFAs EPAStrain Replicates DCW 16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA ERA ETAEPA % DCW Y8406 AVG.2 22.0 2 0 2 4 19 2 3 4 1 2 3 55 12 Y8406U:: AVG.224.6 2 1 2 6 14 1 3 5 1 2 3 55 14 YlLPAAT1 % Ctrl 112 98 153 102 148 7650 120 144 101 109 123 101 113 Y8406U:: AVG.2 21.6 3 1 3 6 14 1 3 4 1 23 57 12 ScLPAATS-5 % Ctrl 98 131 137 125 131 74 56 100 117 86 101 108104 102 Y8406U:: AVG.2 21.4 3 1 3 5 14 1 3 4 1 2 3 58 12 ScLPAATS-6 %Ctrl 97 125 133 121 124 72 52 97 119 88 102 111 106 103

TABLE 24 Desaturase And Elongase Conversion Efficiency In ScLPAATS andYlLPAAT1 Transformant Strains Of Yarrowia lipolytica Y8406 Δ9e Δ8 Δ5 Δ17Strain Replicates Δ12 CE CE CE CE CE Y8406 AVG.2 95 77 92 90 92Y8406U::YlLPAAT1 AVG.2 93 82 92 87 90 % Ctrl 98 107 99 97 98Y8406U::ScLPAATS-5 AVG.2 94 83 93 89 92 % Ctrl 98 108 100 99 100Y8406U::ScLPAATS-6 AVG.2 94 83 93 89 92 % Ctrl 99 109 101 99 100

Based on the data in Table 23 and Table 24 above, overexpression of bothScLPAATS and YlLPAAT1 in EPA strains Y8406U::YlLPAAT1,Y8406U::ScLPAATS-5 and Y8406U::ScLPAATS-6 resulted in reduction (to 76%or below of the control) of the concentration of LA (18:2) as a weight %of TFAs [“LA % TFAs”], and an increase (to at least 7% of the control)in the conversion efficiency of the Δ9 elongase. ScLPAATS and YlLPAAT1have a similar effect on lipid profile.

The results obtained above were then compared to those obtained inExample 4, although different means were utilized to characterize theLPLATs. Specifically, in Example 4, linearized DNA carrying the LPLATswere transformed by chromosomal integration, since the vectors lackedARS sequences. This resulted in stable integrations and the strains weregrown in the relatively rich, non-selective FM growth medium during bothpreculture and 2 days growth prior to being transferred to HGM.

In Example 8, the functional characterization of YlLPAAT1 and ScLPAATSwas done on a replicating plasmid. Thus, Yarrowia lipolytica strainY8406 was transformed with circular DNA carrying each LPAAT and ARSsequence. To maintain these plasmids and assay gene expression withoutintegration, it was necessary to grow the transformants on selectivemedium (i.e., CSM-U medium) during both preculture and 2 days growthprior to being transferred to HGM.

These differences described above can contribute to differences in lipidprofile and content, as illustrated by the expression of YlLPAAT1 inExamples 4 and 8. The change over control in LA % TFAs, EPA % TFAs, andΔ9 elongase conversion efficiency were 63%, 115%, and 115%,respectively, upon expression of YlLPAAT in Example 4, whereas thechange over control in LA % TFAs, EPA % TFAs, and Δ9 elongase conversionefficiency were were 76%, 101%, and 107%, respectively, upon expressionof YlLPAAT in Example 8. Thus, the improvements in Δ9 elongation andLC-PUFA biosynthesis in Example 8 are minimized when compared to thoseobserved in Example 4. These differences can be attributed to the“position effects” of chromosomal integration and/or different growthconditions.

Since the improvements in LC-PUFA biosynthesis (measured as reduction inLA % TFAs, increase in EPA % TFAs and increase in Δ9 elongase conversionefficiency) are similar for both ScLPAATS and YlLPAAT when transformedin Yarrowia lipolytica strain Y8406 on a replicating plasmid, it isanticipated that both LPLAATs will also function similarly when stablyintegrated into the host chromosome. Thus, ScLPAATS will likely improvethe lipid profile in a manner similar to that observed in Examples 4 and5, when YlLPAAT1 was stably integrated into the host chromosome.

What is claimed is:
 1. A recombinant oleaginous microbial host cell forimproved production of at least one long-chain polyunsaturated fattyacid, said host cell comprising at least one isolated polynucleotideencoding a polypeptide having at least acyl-CoA:lysophospholipidacyltransferase activity, wherein the polypeptide comprises (i) at least90% amino acid identity, based on the Clustal W method of alignment,compared to the amino acid sequence of SEQ ID NO:11, and (ii) at leastone membrane bound O-acyltransferase protein family motif selected fromthe group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQID NO:28; wherein the isolated polynucleotide is operably linked to atleast one regulatory sequence, and further wherein the host cell has atleast one improvement selected from the group consisting of: (i) anincrease in C₁₈ to C₂₀ elongation conversion efficiency compared to acontrol host cell, and (ii) an increase in delta-4 desaturationconversion efficiency compared to a control host cell.
 2. Therecombinant host cell of claim 1, wherein the at least one long-chainpolyunsaturated fatty acid is selected from the group consisting of:eicosadienoic acid, dihomo-gamma-linolenic acid, arachidonic acid,eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid,docosatetraenoic acid, omega-6 docosapentaenoic acid, omega-3docosapentaenoic acid and docosahexaenoic acid.
 3. The recombinant hostcell of claim 1, wherein the isolated polynucleotide is stablyintegrated in the host cell; and further wherein the improvement isselected from the group consisting of: a) an increase in C₁₈ to C₂₀elongation conversion efficiency of at least 4% compared to a controlhost cell; and b) an increase in delta-4 desaturation conversionefficiency of at least 5% compared to a control host cell.
 4. Therecombinant host cell of claim 3, wherein the improvement is selectedfrom the group consisting of: a) an increase in C₁₈ to C₂₀ elongationconversion efficiency of at least 13% compared to a control host cell,wherein the recombinant host cell produces eicosapentaenoic acid; b) anincrease in C₁₈ to C₂₀ elongation conversion efficiency of at least 4%compared to a control host cell, wherein the recombinant host cellproduces docosahexaenoic acid; c) an increase in delta-4 desaturationconversion efficiency of at least 18% compared to a control host cell,wherein the recombinant host cell produces docosahexaenoic acid; d) anincrease of at least 9% of eicosapentaenoic acid measured as a weightpercent of the total fatty acids compared to a control host cell; e) anincrease of at least 2% of eicosapentaenoic acid measured as a weightpercent of the total fatty acids compared to a control host cell,wherein the recombinant host cell produces docosahexaenoic acid; and f)an increase of at least 9% of docosahexaenoic acid measured as a weightpercent of the total fatty acids compared to a control host cell.
 5. Therecombinant host cell of claim 1, wherein the host cell is a yeast. 6.The recombinant host cell of claim 5, wherein the yeast is Yarrowialipolytica.
 7. The recombinant host cell of claim 1, wherein thepolypeptide comprises at least 95% amino acid identity compared to theamino acid sequence of SEQ ID NO:11.
 8. The recombinant host cell ofclaim 1, wherein the regulatory sequence comprises a heterologouspromoter.
 9. A method for making an oil comprising eicosapentaenoic acidand/or docosahexaenoic acid comprising: a) culturing the recombinanthost cell of claim 1, wherein an oil comprising eicosapentaenoic acidand/or docosahexaenoic acid is produced; and b) optionally recoveringthe oil of step (a).
 10. The method of claim 9, wherein the recoveredoil of step (b) is further processed.
 11. The method of claim 9, whereinthe host cell is an oleaginous yeast.
 12. The method of claim 11,wherein the oleaginous yeast is Yarrowia lipolytica.